MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Current Protocols in Molecular Biology' source. Thu, 09 Feb 2012 16:56:40 +0100 http://www.medworm.com/index.php?rid=5596553&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22237856%26dopt%3DAbstract Authors: Gallagher SR Abstract One-dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample. Separated proteins can be recovered from polyacrylamide gels for subsequent characterization by a variety of secondary techniques, such as mass spectrometry to determine post-translational modifications and the amino acid sequence. In addition, one-dimensional electrophoresis is the standard first step in immunoblotting and immunodetection. Protein separations in vertical slab gels are performed in a variety of formats. Most recently, small format minigels are typical due to their ease of use, low relative cost, and ready commercial availability. Larger gels provide more separation area and thus better resolution for c... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5596553 Sun, 01 Jan 2012 05:00:00 +0100 5596553 http://www.medworm.com/index.php?rid=5596552&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22237857%26dopt%3DAbstract Authors: Lin JJ, Carey M Abstract In the study of gene regulation, it is often necessary to employ functional assays that investigate the action or mechanism of specific promoters or enhancer-binding factors and their role in transcription by RNA polymerase II. Although many assays measure the transcription of a gene under the control of an endogenous or model activator in vivo, it is often useful to recreate transcription in vitro in order to study specific regulatory mechanisms. In this unit, protocols are presented that will allow the investigator to perform in vitro transcription using preinitiation complexes assembled from cellular extracts on either naked DNA or chromatin templates. Curr. Protoc. Mol. Biol. 97:12.14.1-12.14.19. © 2012 by John Wiley & Sons, Inc. PMID:... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5596552 Sun, 01 Jan 2012 05:00:00 +0100 5596552 http://www.medworm.com/index.php?rid=5596551&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22237858%26dopt%3DAbstract Authors: Xu D Abstract Protein databases have become a crucial part of modern biology. Huge amounts of data for protein structures, functions, and particularly sequences are being generated. Searching databases is often the first step in the study of a new protein. Comparison between proteins or between protein families provides information about the relationship between proteins within a genome or across different species, and hence offers much more information than can be obtained by studying only an isolated protein. In addition, secondary databases derived from experimental databases are also widely available. These databases reorganize and annotate the data or provide predictions. The use of multiple databases often helps researchers understand the structure and function of a ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5596551 Sun, 01 Jan 2012 05:00:00 +0100 5596551 http://www.medworm.com/index.php?rid=5596550&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22237859%26dopt%3DAbstract Authors: Rajarajan K, Engels MC, Wu SM Abstract The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enab... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5596550 Sun, 01 Jan 2012 05:00:00 +0100 5596550 http://www.medworm.com/index.php?rid=5309975&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21987054%26dopt%3DAbstract Authors: Sander JD, Maeder ML, Joung JK Abstract Engineered designer nucleases can be used to efficiently modify genomic sequence in a wide variety of model organisms and cell types. Zinc finger nucleases (ZFNs), consisting of an engineered zinc finger array fused to a non-specific cleavage domain, have been extensively used to modify a broad range of endogenous genes. Protocols for engineering ZFNs targeted to specific gene sequences of interest using the context-dependent assembly (CoDA) method are described in this unit. Curr. Protoc. Mol. Biol. 96:12.13.1-12.13.16. © 2011 by John Wiley & Sons, Inc. PMID: 21987054 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5309975 Sat, 01 Oct 2011 04:00:00 +0100 5309975 http://www.medworm.com/index.php?rid=5309974&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21987055%26dopt%3DAbstract Authors: Rokas A Abstract Phylogenetic analysis is the study of evolutionary relationships among molecules, phenotypes, and organisms. In the context of protein sequence data, phylogenetic analysis is one of the cornerstones of comparative sequence analysis and has many applications in the study of protein evolution and function. This unit provides a brief review of the principles of phylogenetic analysis and describes several different standard phylogenetic analyses of protein sequence data using the RAXML (Randomized Axelerated Maximum Likelihood) Program. Curr. Protoc. Mol. Biol. 96:19.11.1-19.11.14. © 2011 by John Wiley & Sons, Inc. PMID: 21987055 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5309974 Sat, 01 Oct 2011 04:00:00 +0100 5309974 http://www.medworm.com/index.php?rid=5309973&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21987056%26dopt%3DAbstract Authors: Shendure JA, Porreca GJ, Church GM, Gardner AF, Hendrickson CL, Kieleczawa J, Slatko BE Abstract Efficient and cost-effective DNA sequencing technologies are critical to the progress of molecular biology. This overview of DNA sequencing strategies provides a high-level review of seven distinct approaches to DNA sequencing: (a) dideoxy sequencing; (b) solid phase sequencing; (c) sequencing-by-hybridization; (d) mass spectrometry; (e) cyclic array sequencing; (f) microelectrophoresis; and (g) nanopore sequencing. Other platforms currently in development are also briefly described. The primary focus here is on Sanger dideoxy sequencing, which has been the dominant technology since 1977, and on cyclic array strategies, for which several competitive implementations have been de... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5309973 Sat, 01 Oct 2011 04:00:00 +0100 5309973 http://www.medworm.com/index.php?rid=5309972&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21987057%26dopt%3DAbstract "First generation" automated DNA sequencing technology. Curr Protoc Mol Biol. 2011 Oct;Chapter 7:Unit7.2 Authors: Slatko BE, Kieleczawa J, Ju J, Gardner AF, Hendrickson CL, Ausubel FM Abstract Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencin... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5309972 Sat, 01 Oct 2011 04:00:00 +0100 5309972 http://www.medworm.com/index.php?rid=5309971&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21987058%26dopt%3DAbstract Authors: Daigle D, Simen BB, Pochart P Abstract This unit describes a method to convert PCR products (amplicons) flanked by universal M13 primers into a library for use on all 454 Sequencing Systems (454 Life Sciences, a Roche Company). This is especially useful for simultaneous sequencing and analysis of large numbers of amplicons or for the detection of minor variations within the amplified products. The method described here involves preparing a library of DNA with specific primers containing adaptor sequences recognized by the GS Junior System sequencing process. The data from the sequencing run are processed and analyzed by 454 Life Sciences software. This approach allows for multiplexing of a large number of amplicons to streamline processing and analysis. Any pre-existing un... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5309971 Sat, 01 Oct 2011 04:00:00 +0100 5309971 http://www.medworm.com/index.php?rid=5042045&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21732315%26dopt%3DAbstract Authors: Kramer MF This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. miRNAs are 17 to 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least two times the length of either of the specific forward or reverse primers, each typically ∼20 nt in length. Thus, the target minimum length is ≥40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stabl... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5042045 Thu, 30 Jun 2011 23:00:00 +0100 5042045 http://www.medworm.com/index.php?rid=5042044&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21732316%26dopt%3DAbstract Authors: Zachara NE, Vosseller K, Hart GW O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that is implicated in the etiology of type II diabetes and Alzheimer's disease, as well as cardioprotection. This unit covers simple and comprehensive techniques for identifying proteins modified by O-GlcNAc, studying the enzymes that add and remove O-GlcNAc, and mapping O-GlcNAc modification sites. Curr. Protoc. Mol. Biol. 95:17.6.1-17.6.33. © 2011 by John Wiley & Sons, Inc. PMID: 21732316 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5042044 Thu, 30 Jun 2011 23:00:00 +0100 5042044 http://www.medworm.com/index.php?rid=5042043&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21732317%26dopt%3DAbstract Authors: Pardo CE, Darst RP, Nabilsi NH, Delmas AL, Kladde MP Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein-DNA interactions by probing with cytosine-modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite-converted sequences permits assignment of the me... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5042043 Thu, 30 Jun 2011 23:00:00 +0100 5042043 http://www.medworm.com/index.php?rid=5042042&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21732318%26dopt%3DAbstract Authors: Hollenback SM, Lyman S, Cheng J The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a knock-in of the same gene with less effort and shorter time of establishment. The λ-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=5042042 Thu, 30 Jun 2011 23:00:00 +0100 5042042 http://www.medworm.com/index.php?rid=4694242&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472694%26dopt%3DAbstract Authors: Phillips K, de la Peña AH The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer condition... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694242 Thu, 31 Mar 2011 23:00:00 +0100 4694242 http://www.medworm.com/index.php?rid=4694241&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472695%26dopt%3DAbstract Authors: Xiao R, Moore DD DamIP is a new method for studying DNA-protein interaction in vivo. A mutant form of DNA adenine methyltransferase (DamK9A) from E. coli is fused to the protein of interest and expressed. The fusion protein will bind to target binding sites and introduce N(6)-adenine methylation in nearby sites in the genomic DNA. Methylated DNA fragments are enriched by immunopreciptation with an antibody that recognizes N(6)-methyladenine, and can then be used for further analysis, e.g., real-time PCR, microarray, or high-throughput sequencing. This method is simple and does not require protein-DNA crosslinking or a specific antibody to the protein of interest. This unit describes the application of this method for identification of DNA binding sites in vivo. Curr. Protoc. M... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694241 Thu, 31 Mar 2011 23:00:00 +0100 4694241 http://www.medworm.com/index.php?rid=4694240&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472696%26dopt%3DAbstract Authors: Kulkarni MM This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome-wide (microarrays) and targeted (real-time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target-specific, color-coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35- to 50-... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694240 Thu, 31 Mar 2011 23:00:00 +0100 4694240 http://www.medworm.com/index.php?rid=4694239&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472697%26dopt%3DAbstract Authors: Lohman GJ, Tabor S, Nichols NM The DNA ligase enzyme family catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA. This activity can seal nicks in duplex DNA or join double-stranded DNA fragments having either blunt or cohesive ends. DNA ligases are central enzymes in molecular biology, nucleic acid research, and in next-generation sequencing applications. Reaction conditions and applications for T4 DNA ligase, E. coli DNA ligase, and thermostable DNA ligases are described in this unit. These enzymes differ in their cofactor requirements, substrate specificity, and thermal stability. Curr. Protoc. Mol. Biol. 94:3.14.1-3.14.7. © 2011 by John Wiley & Sons, Inc. PMID: 21472697 [PubMed - in process] (Source:... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694239 Thu, 31 Mar 2011 23:00:00 +0100 4694239 http://www.medworm.com/index.php?rid=4694238&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472698%26dopt%3DAbstract Authors: Gibson DG The yeast Saccharomyces cerevisiae has the capacity to take up and assemble dozens of different overlapping DNA molecules in one transformation event. These DNA molecules can be single-stranded oligonucleotides, to produce gene-sized fragments, or double-stranded DNA fragments, to produce molecules up to hundreds of kilobases in length, including complete bacterial genomes. This unit presents protocols for designing the DNA molecules to be assembled, transforming them into yeast, and confirming their assembly. Curr. Protoc. Mol. Biol. 94:3.22.1-3.22.17. © 2011 by John Wiley & Sons, Inc. PMID: 21472698 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694238 Thu, 31 Mar 2011 23:00:00 +0100 4694238 http://www.medworm.com/index.php?rid=4694237&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21472699%26dopt%3DAbstract Authors: Christodoulou DC, Gorham JM, Herman DS, Seidman JG RNA-seq is a method for studying the transcriptome of cells or tissues by massively parallel sequencing of tens of millions of short DNA fragments. However, the broad dynamic range of gene expression levels, which span more than five orders of magnitude, necessitates considerable over-sequencing to characterize low-abundance RNAs at sufficient depth. Here, we describe a method that enables efficient sequencing of low-abundance RNAs by normalizing or reducing the range spanned by the most abundant RNA species to the least abundant RNA species. This normalization is achieved using an approach that was developed for generating expressed sequence tag (EST) libraries that uses the crab duplex-specific nuclease and exploits the kine... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4694237 Thu, 31 Mar 2011 23:00:00 +0100 4694237 http://www.medworm.com/index.php?rid=4395524&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21225635%26dopt%3DAbstract Authors: Gallagher SR Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. Curr. Protoc. Mol. Biol. 93:A.3D.1-A.3D.14. © 2011 by John Wiley & Sons, Inc. PMID: 21225635 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4395524 Sat, 01 Jan 2011 00:00:00 +0100 4395524 http://www.medworm.com/index.php?rid=4395523&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21225636%26dopt%3DAbstract Authors: R Desjardins P, Conklin DS Quantitation of DNA and RNA by absorbance and fluorescence spectroscopy has been a powerful tool in life sciences for decades. Classic methods of nucleic acid quantitation require the filling of devices, such as cuvettes and capillaries, with sample (traditional methodologies are described in APPENDIX 3D). Analysis of microvolume samples has become of paramount importance as more molecular biology techniques yield progressively smaller amounts of isolated sample and require accurate quantitation of nucleic acids with minimal consumption of sample. Advances in photonic technologies have resulted in a pioneering microvolume system that combines fiber optic technology with the inherent physical properties of the sample to dramatically reduce measurement... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4395523 Sat, 01 Jan 2011 00:00:00 +0100 4395523 http://www.medworm.com/index.php?rid=4395522&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21225637%26dopt%3DAbstract Authors: Fan X, Geisberg JV, Wong KH, Jin Y Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. This unit describes a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from the nucleus. Curr. Protoc. Mol. Biol. 93:13.10B.1-13.10B.8. © 2011 by John Wiley & Sons, Inc. PMID: 21225637 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4395522 Sat, 01 Jan 2011 00:00:00 +0100 4395522 http://www.medworm.com/index.php?rid=4395521&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21225638%26dopt%3DAbstract Authors: C Christodoulou D, Gorham JM, Kawana M, Depalma SR, Herman DS, Wakimoto H Deep sequencing analysis of gene expression (DSAGE) measures global gene transcript levels from only 1 to 2 µg total RNA by massively parallel sequencing of cDNA tags. This unit describes the construction of 21-bp cDNA tag libraries appropriate for massively parallel sequencing and analysis of the resulting sequence data. The adapter oligonucleotides used are optimized for sequencing with current Illumina massively parallel sequencers, and a step-by-step implementation of the analysis protocol is described. The expression profiles obtained are highly reproducible, enabling sensitive detection of differences between experimental conditions as well as assessment of the relative transcript abundance of dif... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4395521 Sat, 01 Jan 2011 00:00:00 +0100 4395521 http://www.medworm.com/index.php?rid=4395518&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21225639%26dopt%3DAbstract Authors: Nichols NM Reaction conditions for a variety of endonucleases are detailed in this unit along with discussions of potential applications. Enzymes covered include BAL 31 nuclease, S1 nuclease, mung bean nuclease, micrococcal nuclease, and DNase I. A general discussion regarding the use of endonucleases to generate nonspecific breaks in dsDNA is also provided. For a detailed discussion of the endonucleases more typically associated with DNA damage repair (e.g., Endo III, IV, V and VIII of E. coli and human APE1), see UNIT 3.9. Curr. Protoc. Mol. Biol. 93:3.12.1-3.12.7. © 2011 by John Wiley & Sons, Inc. PMID: 21225639 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4395518 Sat, 01 Jan 2011 00:00:00 +0100 4395518 http://www.medworm.com/index.php?rid=4040939&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890899%26dopt%3DAbstract Authors: Smith JA Inhibitors of specific cellular pathways are useful for investigating the roles of proteins of unknown function, and for selectively inhibiting a protein in complex pathways to uncover its relationships to other proteins in this and other interacting pathways. This appendix provides links to Web sites that describe cellular processes and pathways along with the various classes of inhibitors, numerous references, downloadable diagrams, and technical tips. Curr. Protoc. Mol. Biol. 92:A.6.1-A.6.19. © 2010 by John Wiley & Sons, Inc. PMID: 20890899 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040939 Thu, 30 Sep 2010 23:00:00 +0100 4040939 http://www.medworm.com/index.php?rid=4040938&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890900%26dopt%3DAbstract Authors: Mayan MD Budding yeast are not permeable to many drugs. This unit provides a protocol in which polygodial is used to permeabilize the cell membrane, thereby allowing budding yeast cells to be treated with drugs that otherwise would be ineffective. Curr. Protoc. Mol. Biol. 92:13.2B.1-13.2B.4. © 2010 by John Wiley & Sons, Inc. PMID: 20890900 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040938 Thu, 30 Sep 2010 23:00:00 +0100 4040938 http://www.medworm.com/index.php?rid=4040937&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890901%26dopt%3DAbstract Authors: Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N With the advent of digital cameras and motorization of mechanical components, computer control of microscopes has become increasingly important. Software for microscope image acquisition should not only be easy to use, but also enable and encourage novel approaches. The open-source software package µManager aims to fulfill those goals. This unit provides step-by-step protocols describing how to get started working with µManager, as well as some starting points for advanced use of the software. Curr. Protoc. Mol. Biol. 92:14.20.1-14.20.17. © 2010 by John Wiley & Sons, Inc. PMID: 20890901 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040937 Thu, 30 Sep 2010 23:00:00 +0100 4040937 http://www.medworm.com/index.php?rid=4040936&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890902%26dopt%3DAbstract Authors: McAvoy T, Nairn AC Methods for assaying serine/threonine protein phosphatases are discussed. Three commonly used protocols are presented that employ either colorimetric or radiometric assays. These methods can be used to assay a variety of preparations of serine/threonine phosphatases, from crude lysates to purified proteins. Strategies for the application of a particular protocol for a particular purpose are discussed. The assays can be used in either a high-throughput mode where simple comparison of activities can be done, or in specific assays where kinetic data can be derived. Curr. Protoc. Mol. Biol. 92:18.18.1-18.18.11. © 2010 by John Wiley & Sons, Inc. PMID: 20890902 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040936 Thu, 30 Sep 2010 23:00:00 +0100 4040936 http://www.medworm.com/index.php?rid=4040935&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890903%26dopt%3DAbstract Authors: He A, Pu WT Recent development of methods for genome-wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. ChIP requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions.... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040935 Thu, 30 Sep 2010 23:00:00 +0100 4040935 http://www.medworm.com/index.php?rid=4040934&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890904%26dopt%3DAbstract Authors: Thompson JF, Steinmann KE Helicos™ Single Molecule Sequencing (SMS) provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an unbiased manner, providing both accurate quantitation and sequence information. Sample preparation does not require ligation or PCR amplification, avoiding the GC-content and size biases observed in other technologies. DNA is simply sheared, tailed with poly(A), and hybridized to a flow cell surface containing oligo(dT) for sequencing-by-synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. Gene expression measurements can be done using first-strand cDNA-based methods (RNA-Seq) or using a novel approach that allows direct hybridization and sequenci... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040934 Thu, 30 Sep 2010 23:00:00 +0100 4040934 http://www.medworm.com/index.php?rid=4040933&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20890905%26dopt%3DAbstract Authors: Potter H, Heller R Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination. Also described are modifications for preparation and transfection of plant protoplasts. Curr. Protoc. Mol. Biol. 92:9.3.1-9.3.10. © 2010 by John Wiley & Sons, Inc. PMID: 20890905 [PubMed - in process] (Source: ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=4040933 Thu, 30 Sep 2010 23:00:00 +0100 4040933 http://www.medworm.com/index.php?rid=3707675&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583094%26dopt%3DAbstract Authors: Chen C, Turk BE Protein kinases vary substantially in their consensus phosphorylation motifs, the residues that are either preferred or deselected by the kinase at specific positions surrounding the phosphorylation site. The protocol described here is used to rapidly determine phosphorylation motifs for serine-threonine kinases. The procedure involves screening an arrayed combinatorial peptide library consisting of 198 biotinylated substrates. Peptides are phosphorylated by the kinase of interest in the presence of radiolabeled ATP and then captured on streptavidin membrane. The membrane is subsequently washed, dried, and exposed to a phosphor screen to visualize and quantify incorporation of radiolabel into the peptides. The phosphorylation motif is thereby derived from the r... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707675 Wed, 30 Jun 2010 03:27:21 +0100 3707675 http://www.medworm.com/index.php?rid=3707674&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583095%26dopt%3DAbstract Authors: Depry C, Zhang J Genetically encodable FRET-based kinase activity reporters (KARs) enable real-time monitoring of kinase activity dynamics in living cells with high spatiotemporal resolution. This unit describes a general protocol for utilizing KARs to visualize kinase activity in living mammalian cells with fluorescence microscopy. PMID: 20583095 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707674 Wed, 30 Jun 2010 03:27:18 +0100 3707674 http://www.medworm.com/index.php?rid=3707673&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583096%26dopt%3DAbstract Authors: Mercan F, Bennett AM Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases. PMID: 20583096 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707673 Wed, 30 Jun 2010 03:27:15 +0100 3707673 http://www.medworm.com/index.php?rid=3707672&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583097%26dopt%3DAbstract Authors: Devkota AK, Kaoud TS, Warthaka M, Dalby KN Protein kinases are enzymes that regulate many cellular events in eukaryotic cells, such as cell-cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site, as well as one or more substrate-binding site(s) that exhibit recognition features for a protein substrate. Thus, by bringing ATP and a substrate into close proximity, each protein kinase can modify its substrate by transferring the gamma phosphate of the ATP molecule to a serine, threonine, or tyrosine residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated, dependent on the phosphorylated versus dephosphorylated forms of the substrate. This unit describes an assay employin... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707672 Wed, 30 Jun 2010 03:27:12 +0100 3707672 http://www.medworm.com/index.php?rid=3707671&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583098%26dopt%3DAbstract Authors: Raha D, Hong M, Snyder M This unit describes ChIP-Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq), and enables the genome-wide identification of binding sites of transcription factors (TFs) and other DNA-binding proteins. The process is initiated by cross-linking DNA and DNA-bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA-binding protein is then used to immunoprecipitate specific DNA-TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30- to 35-nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites. PM... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707671 Wed, 30 Jun 2010 03:27:09 +0100 3707671 http://www.medworm.com/index.php?rid=3707670&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20583099%26dopt%3DAbstract Authors: Darst RP, Pardo CE, Ai L, Brown KD, Kladde MP Exact positions of 5-methylcytosine (m(5)C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which are then converted to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single-nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed. PMID: 20583099 [PubMed - in pr... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3707670 Wed, 30 Jun 2010 03:27:06 +0100 3707670 http://www.medworm.com/index.php?rid=3449675&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20373499%26dopt%3DAbstract Authors: Tian Y, Zhang H This unit describes a method of isolating of proteins from formalin-fixed and paraffin-embedded (FFPE) tissue for mass spectrometry analysis. Heat-induced antigen retrieval is the basis of the protein extraction strategy presented in this protocol. This protocol may be used to identify nuclear, cytosolic, and membrane proteins from FFPE tissues extracted from tissue blocks or slides. Curr. Protoc. Mol. Biol. 90:10.26.1-10.26.7. (c) 2010 by John Wiley & Sons, Inc. PMID: 20373499 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3449675 Wed, 31 Mar 2010 23:00:00 +0100 3449675 http://www.medworm.com/index.php?rid=3449674&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20373500%26dopt%3DAbstract Authors: Geoui T, Urlaub H, Plessmann U, Porschewski P This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the trypt... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3449674 Wed, 31 Mar 2010 23:00:00 +0100 3449674 http://www.medworm.com/index.php?rid=3449673&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20373501%26dopt%3DAbstract Authors: Simon MD Chromatin structure is influenced by post-translational modifications on histones, the principal basic protein component of chromatin. In order to study one of these modifications, lysine methylation, in the context of reconstituted chromatin, this unit describes the installation of analogs of methyl lysine residues into recombinant histones. The modification site is specified by mutating the lysine of interest to cysteine. The mutant histones are expressed and purified, and the cysteine residue alkylated to produce N-methyl aminoethylcysteine, an isosteric analog of methyl lysine. Using different alkylating reagents, it is possible to install analogs of mono-, di-, or trimethyl lysine. While these analogs are not identical to methyl lysine residues, they show similar... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3449673 Wed, 31 Mar 2010 23:00:00 +0100 3449673 http://www.medworm.com/index.php?rid=3449672&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20373502%26dopt%3DAbstract Authors: Vinayavekhin N, Saghatelian A Along with genes and proteins, metabolites play important roles in sustaining life. Their functions include "primary" functions in metabolism and energy storage, as well as "secondary" functions in cell-to-cell signaling, metal acquisition, and virulence. There remains much to be learned about the in vivo roles of metabolites. Approaches that accelerate measurement of metabolite levels directly from cells and tissues should increase our understanding of the diverse roles of metabolites and potentially lead to discovery of novel metabolites and metabolic pathways. Metabolomics is an important comparative tool to study global metabolite levels in samples under various conditions. In this unit, the steps needed to perform a mass spectrometry (MS)-bas... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3449672 Wed, 31 Mar 2010 23:00:00 +0100 3449672 http://www.medworm.com/index.php?rid=3449671&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20373503%26dopt%3DAbstract Authors: Bogdanov EA, Shagina I, Barsova EV, Kelmanson I, Shagin DA, Lukyanov SA The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. Curr. Protoc. Mol. Biol. 90:5.12.1-5.12.27. (c) 2010 by John Wiley & Sons, Inc. ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3449671 Wed, 31 Mar 2010 23:00:00 +0100 3449671 http://www.medworm.com/index.php?rid=3172352&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069534%26dopt%3DAbstract Authors: Freeze HH, Kranz C Nearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space. Many glycans can be attached to proteins, but the most common are the N-linked glycans (oligosaccharides). These chains are added very soon after a protein enters the ER, but they undergo extensive remodeling (processing), especially in the Golgi. Processing changes the sensitivity of the N-glycan to enzymes that cleave entire sugar chains or individual monosaccharides, which also changes the migration of the protein on SDS gels. These changes can be used to indicate when a protein has passed a particular subcellular location. This unit details some of the methods used to track a prote... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172352 Fri, 01 Jan 2010 00:00:00 +0100 3172352 http://www.medworm.com/index.php?rid=3172351&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069535%26dopt%3DAbstract Authors: Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, Nekrutenko A, Taylor J High-throughput data production has revolutionized molecular biology. However, massive increases in data generation capacity require analysis approaches that are more sophisticated, and often very computationally intensive. Thus, making sense of high-throughput data requires informatics support. Galaxy (http://galaxyproject.org) is a software system that provides this support through a framework that gives experimentalists simple interfaces to powerful tools, while automatically managing the computational details. Galaxy is distributed both as a publicly available Web service, which provides tools for the analysis of genomic, comparative genomic, and functional genomic data, or a downl... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172351 Fri, 01 Jan 2010 00:00:00 +0100 3172351 http://www.medworm.com/index.php?rid=3172350&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069536%26dopt%3DAbstract Authors: Fullwood MJ, Han Y, Wei CL, Ruan X, Ruan Y Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cells are treated with formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172350 Fri, 01 Jan 2010 00:00:00 +0100 3172350 http://www.medworm.com/index.php?rid=3172349&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069537%26dopt%3DAbstract Authors: Tiwari VK, Baylin SB Understanding of transcriptional regulation has advanced in recent years in part due to development of technologies which allow determination of physical proximities between interacting chromatin regions at a resolution beyond that offered by conventional microscopy techniques. However, these methods do not specifically identify the protein component(s) that might mediate such interactions. This unit provides a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to simultaneously identify physical proximities between chromatin elements as well as the proteins that mediate these interactions. The unit further explores how the 6C assay can be combined with other techniques for a complete, cell-type-specific mapp... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172349 Fri, 01 Jan 2010 00:00:00 +0100 3172349 http://www.medworm.com/index.php?rid=3172348&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069538%26dopt%3DAbstract Authors: Miranda TB, Kelly TK, Bouazoune K, Jones PA Methylation-sensitive single-molecule analysis of chromatin structure is a high-resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro-remodeled nucleosomes by treatment with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease-based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population. PMID: 20069538 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172348 Fri, 01 Jan 2010 00:00:00 +0100 3172348 http://www.medworm.com/index.php?rid=3172347&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20069539%26dopt%3DAbstract Authors: Nagalakshmi U, Waern K, Snyder M A recently developed technique called RNA Sequencing (RNA-Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger sequencing- and microarray-based methods. In the RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next-generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole-genome transcriptome map. RNA-Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5' and 3' ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA-Seq using t... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=3172347 Fri, 01 Jan 2010 00:00:00 +0100 3172347 http://www.medworm.com/index.php?rid=2877246&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19816929%26dopt%3DAbstract Authors: Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be con... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2877246 Wed, 30 Sep 2009 23:00:00 +0100 2877246 http://www.medworm.com/index.php?rid=2877245&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19816930%26dopt%3DAbstract Authors: Ashrafi EH, Paul N Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3' end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymeras... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2877245 Wed, 30 Sep 2009 23:00:00 +0100 2877245 http://www.medworm.com/index.php?rid=2877244&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19816931%26dopt%3DAbstract Authors: Mangan ME, Williams JM, Kuhn RM, Lathe WC Electronic data resources can enable molecular biologists to query and display many useful features that make benchwork more efficient and drive new discoveries. The UCSC Genome Browser provides a wealth of data and tools that advance one's understanding of genomic context for many species, enable detailed understanding of data, and provide the ability to interrogate regions of interest. Researchers can also supplement the standard display with their own data to query and share with others. Effective use of these resources has become crucial to biological research today, and this unit describes some practical applications of the UCSC Genome Browser. PMID: 19816931 [PubMed - in process] (Source: Current Protocols in Molecular Biolog... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2877244 Wed, 30 Sep 2009 23:00:00 +0100 2877244 http://www.medworm.com/index.php?rid=2877243&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19816932%26dopt%3DAbstract Authors: Hall B, Micheletti JM, Satya P, Ogle K, Pollard J, Ellington AD Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions. PMID: 19816932 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2877243 Wed, 30 Sep 2009 23:00:00 +0100 2877243 http://www.medworm.com/index.php?rid=2877242&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19816933%26dopt%3DAbstract Authors: Hall B, Arshad S, Seo K, Bowman C, Corley M, Jhaveri SD, Ellington AD This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. PMID: 19816933 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2877242 Wed, 30 Sep 2009 23:00:00 +0100 2877242 http://www.medworm.com/index.php?rid=2573187&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19575475%26dopt%3DAbstract Authors: Smith JA Inhibitors of specific cellular pathways are useful for investigating the roles of proteins of unknown function, and for selectively inhibiting a protein in complex pathways to uncover its relationships to other proteins in this and other interacting pathways. This appendix provides links to Web sites that describe cellular processes and pathways along with the various classes of inhibitors, numerous references, downloadable diagrams, and technical tips. Curr. Protoc. Mol. Biol. 87:A.6.1-A.6.5. (c) 2009 by John Wiley & Sons, Inc. PMID: 19575475 [PubMed - as supplied by publisher] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2573187 Tue, 30 Jun 2009 23:00:00 +0100 2573187 http://www.medworm.com/index.php?rid=2573186&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19575476%26dopt%3DAbstract Authors: Diaz S, Varki A Useful information about glycoconjugates can be obtained by labeling their aglycone (noncarbohydrate) portions-e.g., labeling proteins with radioactive amino acids-and then using techniques described elsewhere in this chapter to infer the presence, type, and nature of glycan chains. This unit describes metabolic labeling techniques that provide more specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Following metabolic labeling, the radioactive glycoconjugate of interest is isolated, individual glycosylation sites are identified and separated if necessary, and the labeled glycans are subjected to structural analysis. Curr. Protoc. Mol. Biol. 87:17.4.1-17.4.15. (c) 2009 by John Wiley & Sons, Inc. ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2573186 Tue, 30 Jun 2009 23:00:00 +0100 2573186 http://www.medworm.com/index.php?rid=2573185&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19575477%26dopt%3DAbstract Authors: Bell GW, Lewitter F In the past fifteen years, new classes of regulatory RNAs have been discovered, previously hidden in the transcriptome mostly due to their small size. These small regulatory RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Numerous databases have been developed to store information about these small regulatory RNAs, and many tools have been developed to work with the data. This overview introduces the reader to the many resources available for working with small regulatory RNAs. Curr. Protoc. Mol. Biol. 87:19.8.1-19.8.13. (c) 2009 by John Wiley & Sons, Inc. PMID: 19575477 [PubMed - as supplied by publisher] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2573185 Tue, 30 Jun 2009 23:00:00 +0100 2573185 http://www.medworm.com/index.php?rid=2573184&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19575478%26dopt%3DAbstract Authors: Davidson EA, Dlugosz PJ, Levy M, Ellington AD This unit describes a protocol for the directed evolution of proteins utilizing in vitro compartmentalization. This method uses a large number of independent in vitro transcription and translation (IVTT) reactions in water droplets suspended in an oil emulsion to enable selection of proteins that bind a target molecule. Protein variants that bind the target also bind to and allow recovery of the genes that encoded them. This protocol serves as a basis for carrying out selections in emulsions, and can potentially be modified to select for other functionalities, including catalysis. This selection method is advantageous compared to alternative selection protocols due to the ability to screen through very large-size libraries and the ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2573184 Tue, 30 Jun 2009 23:00:00 +0100 2573184 http://www.medworm.com/index.php?rid=2573183&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19575479%26dopt%3DAbstract Authors: Scanlon MJ, Ohtsu K, Timmermans MC, Schnable PS Protocols for laser microdissection and linear amplification of RNA from fixed, sectioned plant tissues are described. When combined with quantitative RT-PCR, microarray analysis, or RNA-sequencing, these procedures enable quantitative analyses of transcript accumulation from microscopic quantities of specific plant organs, tissues, or single cells. Curr. Protoc. Mol. Biol. 87:25A.3.1-25A.3.15 (c) 2009 by John Wiley & Sons, Inc. PMID: 19575479 [PubMed - as supplied by publisher] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2573183 Tue, 30 Jun 2009 23:00:00 +0100 2573183 http://www.medworm.com/index.php?rid=2326636&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19343706%26dopt%3DAbstract Authors: Urbach JM, Wei T, Liberati N, Grenfell-Lee D, Villanueva J, Wu G, Ausubel FM PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software. PMID: 19343706 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2326636 Wed, 01 Apr 2009 04:00:00 +0100 2326636 http://www.medworm.com/index.php?rid=2326635&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19343707%26dopt%3DAbstract Authors: Kotova E, Coleman T, Serebriiskii I The yeast two-hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein-protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA-binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA-binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain-fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also exp... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2326635 Wed, 01 Apr 2009 04:00:00 +0100 2326635 http://www.medworm.com/index.php?rid=2326634&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19343708%26dopt%3DAbstract Authors: Bitinaite J, Nichols NM This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single-stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment. Two b... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2326634 Wed, 01 Apr 2009 04:00:00 +0100 2326634 http://www.medworm.com/index.php?rid=2326628&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19343709%26dopt%3DAbstract Authors: Mortensen RM, Kingston RE To determine the function of a gene in vitro, expression in heterologous cells is often employed. This can be done by transient expression, but often requires a more permanent expression of the gene and the creation of a cell line. This process can involve decisions as to the nature of construct used for expression, and invariably uses some strategy to select the transfected cells. Typically, these strategies use one of a number of genes that confer resistance to an added drug that will kill untransfected cells but not the transfected cells (positive selection). Alternatively, sometimes the strategy uses a gene that will confer sensitivity to a compound and kills the transfected cells (negative selection). This chapter discusses some of the strategies... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2326628 Wed, 01 Apr 2009 04:00:00 +0100 2326628 http://www.medworm.com/index.php?rid=2135528&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19170025%26dopt%3DAbstract Authors: This appendix lists the full contact information, including website URLs, for all suppliers mentioned in Current Protocols in Molecular Biology. PMID: 19170025 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2135528 Thu, 01 Jan 2009 05:00:00 +0100 2135528 http://www.medworm.com/index.php?rid=2135527&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19170026%26dopt%3DAbstract Authors: Sasse J, Gallagher SR This unit describes protocols for detecting protein in a gel by Coomassie blue, silver, or fluorescent staining. As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Alternate protocols describe rapid Coomassie and silver staining methods, as well as fluorescent stains that are specific for phosphoproteins and glycoproteins. Staining of proteins in SDS-polyacrylamide gels is described; variations for fluorescent staining of ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2135527 Thu, 01 Jan 2009 05:00:00 +0100 2135527 http://www.medworm.com/index.php?rid=2135526&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19170027%26dopt%3DAbstract Authors: Katagiri F, Glazebrook J DNA microarray technology allows simultaneous measurement of the mRNA levels of thousands of genes. This powerful technology has applications in addressing many biological questions that were not approachable previously; however, the enormous size of microarray data sets leads to issues of experimental design and statistical analysis that are unfamiliar to many molecular biologists. The type of array used, the design of the biological experiment, the number of experimental replicates, and the statistical method for data analysis should all be chosen based on the scientific goals of the investigator. This overview presents a discussion of the relative merits and limitations of various methods with respect to some common applications of microarray experi... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2135526 Thu, 01 Jan 2009 05:00:00 +0100 2135526 http://www.medworm.com/index.php?rid=2135525&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19170028%26dopt%3DAbstract Authors: Katagiri F, Glazebrook J In expression profiling studies, it is often necessary to identify groups of genes with similar expression profiles in a variety of samples, and/or groups of samples with similar expression profiles. Each profile can be expressed as a single data point in a space with the same number of dimensions as there are parameters in the profiles. In this way, pattern discovery among expression profiles is translated into pattern discovery in the spatial distribution of data points: the similarity between profiles is defined by the distance between the corresponding data points. Various multivariate analysis methods, such as clustering and dimensionality reduction methods, are used to summarize the data point distribution to help the investigator recognize major... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2135525 Thu, 01 Jan 2009 05:00:00 +0100 2135525 http://www.medworm.com/index.php?rid=2135524&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19170029%26dopt%3DAbstract Authors: Bouvier J, Cheng JG Gene targeting in the mouse is an essential tool for studying gene function and creating models of human disease. The method described in this unit takes advantage of bacterial artificial chromosomes, Cre/loxP and FLPe/FRT systems, and recently evolved recombineering approaches to simplify the preparation of targeting constructs for generation of conditional knockout (CKO) animals. This method has been used to generate >30 CKO constructs, most of them successfully used to target mouse ES cells and establish mutant mice. Design and preparation of the CKO construct, as well as step-wise troubleshooting guidelines, are described in detail. Curr. Protoc. Mol. Biol. 85:23.13.1-23.13.27. (c) 2009 by John Wiley & Sons, Inc. PMID: 19170029 [PubMed - in p... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=2135524 Thu, 01 Jan 2009 05:00:00 +0100 2135524 http://www.medworm.com/index.php?rid=1922173&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972381%26dopt%3DAbstract Authors: Sasse J, Gallagher SR The staining of proteins bound to a transfer membrane can be useful for determining the efficiency of transfer and marking the location of molecular weight standards. This unit describes three methods for staining blots, using India ink, gold labeling, and fluorescent labeling with SYPRO Ruby. Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for enhancement of staining using alkali treatment. Curr. Protoc. Mol. Biol. 84:10.7.1-10.7.6. (c) 2008 by John Wiley & Sons, Inc. PMID: 18972381 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922173 Wed, 01 Oct 2008 04:00:00 +0100 1922173 http://www.medworm.com/index.php?rid=1922172&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972382%26dopt%3DAbstract Authors: Chernomoretz A, Bush A, Yu R, Gordon A, Colman-Lerner A This unit describes a method for quantifying various cellular parameters (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a p... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922172 Wed, 01 Oct 2008 04:00:00 +0100 1922172 http://www.medworm.com/index.php?rid=1922171&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972383%26dopt%3DAbstract This article describes a wide range of useful visualization tools designed to better enable discrimination of different features in multilabeled tissue or cell samples. These commercially available tools can be attached to the standard laboratory light microscope to significantly enhance the power of light microscopy. Curr. Protoc. Mol. Biol. 84:14.19.1-14.19.15. (c) 2008 by John Wiley & Sons, Inc. PMID: 18972383 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922171 Wed, 01 Oct 2008 04:00:00 +0100 1922171 http://www.medworm.com/index.php?rid=1922170&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972384%26dopt%3DAbstract Authors: Feige JN, Lagouge M, Auwerx J The maintenance of metabolic homeostasis relies on the balanced intake of nutrients from food. Consequently, diet composition strongly impacts whole-body physiology. Dietary formulations with strong nutrient imbalances can lead to metabolic disorders, with lipids and simple sugars playing a prominent role. This unit describes how diet formulation can be modified to generate mouse models of human metabolic pathologies, and it details methodological procedures linked to dietary manipulations, including caloric restriction and introduction of a test compound. Curr. Protoc. Mol. Biol. 84:29B.5.1-29B.5.12. (c) 2008 by John Wiley & Sons, Inc. PMID: 18972384 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922170 Wed, 01 Oct 2008 04:00:00 +0100 1922170 http://www.medworm.com/index.php?rid=1922169&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972385%26dopt%3DAbstract Authors: Nichols NM, Yue D Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed. PMID: 18972385 [PubMed - in process] (Source: Current Protocols ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922169 Wed, 01 Oct 2008 04:00:00 +0100 1922169 http://www.medworm.com/index.php?rid=1922168&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972386%26dopt%3DAbstract Authors: Nichols NM, Tabor S, McReynolds LA T4 RNA ligase 1 catalyzes the ATP-dependent covalent joining of single-stranded 5'-phosphoryl termini of DNA or RNA to single-stranded 3'-hydroxyl termini of DNA or RNA. T4 RNA ligase 2 also catalyzes the joining of a 3'-hydroxyl terminus of RNA to a 5'-phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double-stranded substrates. A truncated form of T4 RNA ligase 2 requires a pre-adenylated substrate for ligation. This unit describes specific reaction conditions, as well as applications such as radioactive labeling of the 3' termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning. PMID: 18972386 [P... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922168 Wed, 01 Oct 2008 04:00:00 +0100 1922168 http://www.medworm.com/index.php?rid=1922167&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972387%26dopt%3DAbstract Authors: Kucera RB, Nichols NM This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion-bypass DNA polymerases that play a role in DNA damage repair. PMID: 18972387 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922167 Wed, 01 Oct 2008 04:00:00 +0100 1922167 http://www.medworm.com/index.php?rid=1922166&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972388%26dopt%3DAbstract Authors: Yue D, Tabor S, Nichols NM Terminal deoxynucleotidyl transferase (TdT), is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides at the 3'-hydroxyl terminus of DNA, accompanied by the release of inorganic phosphate. TdT does not require a template and will not copy one. Reaction conditions and some applications are described in this unit, including cloning DNA fragments, labeling the 3' terminus of DNA with (32)P or nonradioactive tags, synthesizing model polydeoxynucleotide homopolymers, and detecting DNA damage and apoptotic cells. PMID: 18972388 [PubMed - in process] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922166 Wed, 01 Oct 2008 04:00:00 +0100 1922166 http://www.medworm.com/index.php?rid=1922165&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972389%26dopt%3DAbstract Authors: Tzertzinis G, Tabor S, Nichols NM Reverse transcriptases (RTs) are multifunctional enzymes, but are mainly used as RNA-directed DNA polymerases in first-strand cDNA synthesis. Specifically, oligodeoxynucleotides are used as primers for extension on RNA templates. The DNA synthesized from an RNA template is referred to as complementary DNA (cDNA) and is often used as a template for PCR or converted to dsDNA for cloning. This unit describes appropriate reaction conditions for RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV), along with applications such as synthesizing cDNA, 3' fill-in reactions, and labeling the 3' terminus of DNA fragments with 5' protruding ends, and DNA sequencing. PMID: 18972389 [PubMed - in process] (Source: Current Pr... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922165 Wed, 01 Oct 2008 04:00:00 +0100 1922165 http://www.medworm.com/index.php?rid=1922164&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972390%26dopt%3DAbstract Authors: Paschal BM, McReynolds LA, Noren CJ, Nichols NM This unit describes DNA-dependent, RNA-dependent, and template-independent RNA polymerases. DNA-dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA-dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template-independent polymerase, catalyzes the incorporation of AMP residues onto the free 3'-h... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922164 Wed, 01 Oct 2008 04:00:00 +0100 1922164 http://www.medworm.com/index.php?rid=1922163&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18972391%26dopt%3DAbstract Authors: Evans TC, Nichols NM In vivo DNA damage impacts the genetic stability of an organism; therefore, multiple pathways utilizing a large number of enzymes have evolved to repair DNA damage. This unit focuses on enzymes involved in base excision repair (BER). The BER enzymes possessing N-glycosylase activity can find and remove a wide variety of damaged bases in a sea of normal bases. The combination of unique substrate specificity, accuracy, and robust in vitro activity of many of these enzymes has led to their use in various experimental techniques, including site-specific DNA cleavage. The enzymes described in this unit are active on many substrates including oxidized purines and pyrimidines, alkylated bases, abasic sites, pyrimidine dimers, deaminated cytosines, and deaminated ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1922163 Wed, 01 Oct 2008 04:00:00 +0100 1922163 http://www.medworm.com/index.php?rid=1817989&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18633991%26dopt%3DAbstract Authors: Gallagher S, Winston SE, Fuller SA, Hurrell JG Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horser... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817989 Tue, 01 Jul 2008 04:00:00 +0100 1817989 http://www.medworm.com/index.php?rid=1817988&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18633992%26dopt%3DAbstract Authors: Yokoyama WM This unit details methods for production of monoclonal antibodies. Two methods are given for production of hybridoma supernatants, including one for large-scale production. A protocol for large-scale production of hybridomas or cell lines is presented for use in isolation of cellular proteins. Finally, a method is given for producing and obtaining mouse ascites fluid containing monoclonal antibody. Recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council) on the use of animals for producing monoclonal antibodies are also discussed. These recommendations are designed to foster the judicious use of animals and to strongly encourage the use of in vitro methods whenever ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817988 Tue, 01 Jul 2008 04:00:00 +0100 1817988 http://www.medworm.com/index.php?rid=1817987&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18633993%26dopt%3DAbstract Authors: Moulton JD, Yan YL Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and maturation of miRNA, although they can also inhibit other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817987 Tue, 01 Jul 2008 04:00:00 +0100 1817987 http://www.medworm.com/index.php?rid=1817996&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425759%26dopt%3DAbstract Authors: Treco DA, Winston F Yeast cultures can be grown, maintained, and stored in liquid media or on agar plates using techniques similar to those for bacterial cultures. This unit describes culture conditions for these basic techniques. Additional methods describe determination of yeast mating type, diploid construction, sporulation, tetrad dissection, and random spore analysis. PMID: 18425759 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817996 Tue, 01 Apr 2008 04:00:00 +0100 1817996 http://www.medworm.com/index.php?rid=1817995&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425760%26dopt%3DAbstract Authors: Winston F Many fundamental biological processes have been elucidated by the isolation and analysis of mutants that are defective in such processes. Therefore, the methods to generate mutants are of great importance in model organisms. This unit describes two protocols for mutagenesis of yeast-using ethyl methanesulfate (EMS) and ultraviolet (UV) light. Each of these methods has been used successfully for many years. PMID: 18425760 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817995 Tue, 01 Apr 2008 04:00:00 +0100 1817995 http://www.medworm.com/index.php?rid=1817994&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425761%26dopt%3DAbstract Authors: Vokes MS, Carpenter AE Visual analysis is required to perform many biological experiments, from counting yeast colonies to measuring the size and shape of individual cells or the intensity of fluorescently labeled proteins within them. This unit outlines the use of CellProfiler, a free, open-source image analysis tool that extracts quantitative information from biological images. It includes a step-by-step protocol for automated analysis of the number, color, and size of yeast colonies growing on agar plates, but the methods can be adapted to identify and measure any objects in images. The flexibility of the software allows users to tailor pipelines of adjustable modules to fit different biological experiments, to generate accurate measurements from dozens or even hundreds of ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817994 Tue, 01 Apr 2008 04:00:00 +0100 1817994 http://www.medworm.com/index.php?rid=1817993&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425762%26dopt%3DAbstract Authors: Bagasra O This unit provides detailed methods and material descriptions for in situ hybridization following in situ amplification of DNA or RNA by PCR. It includes all essential components of the techniques, including variations suitable for different kinds of tissue and cell preparations. Planning, controls, and critical parameters for the amplification steps are discussed. PMID: 18425762 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817993 Tue, 01 Apr 2008 04:00:00 +0100 1817993 http://www.medworm.com/index.php?rid=1817992&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425763%26dopt%3DAbstract Authors: Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins. PMID: 18425763 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817992 Tue, 01 Apr 2008 04:00:00 +0100 1817992 http://www.medworm.com/index.php?rid=1817991&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425764%26dopt%3DAbstract Authors: Mortensen R Gene targeting by homologous recombination is a powerful and widely used technique for introduction of specific gene mutations (frequently a gene inactivation) in transgenic animals. The basic method detailed in this unit uses sequences homologous to the endogenous gene flanking the mutation. While methods using bacterial artificial chromosomes (BACs) and recombineering may be used, in most cases simpler bacterial plasmid clones with several kb of homology are sufficient. This protocol details the strategic factors in designing the constructs for selection and screening for homologous recombination. PMID: 18425764 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817991 Tue, 01 Apr 2008 04:00:00 +0100 1817991 http://www.medworm.com/index.php?rid=1817990&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18425765%26dopt%3DAbstract Authors: Mortensen R Under some circumstances, it may be desirable to produce a mouse cell clone in which both alleles of a desired gene are mutated. This may be because the mutation causes embryonic lethality in homozygous animals, or to test cultured cells before an animal is produced. This protocol details an easy method for obtaining homozygous cells by homologous recombination without the need for two targeting events. PMID: 18425765 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817990 Tue, 01 Apr 2008 04:00:00 +0100 1817990 http://www.medworm.com/index.php?rid=1818004&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265398%26dopt%3DAbstract Authors: Dostie J, Zhan Y, Dekker J Chromosome conformation capture (3C) is used to quantify physical DNA contacts in vivo at high resolution. 3C was first used in yeast to map the spatial chromatin organization of chromosome III, and in higher eukaryotes to demonstrate that genomic DNA elements regulate target genes by physically interacting with them. 3C has been widely adopted for small-scale analysis of functional chromatin interactions along (cis) or between (trans) chromosomes. For larger-scale applications, chromosome conformation capture carbon copy (5C) combines 3C with ligation-mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra-high-throughput DNA sequencing. 5C allows the mapping of extensive networks... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818004 Mon, 01 Oct 2007 04:00:00 +0100 1818004 http://www.medworm.com/index.php?rid=1818003&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265399%26dopt%3DAbstract Authors: D Ellington A This unit provides a brief description of the different approaches that can be used to identify functional peptides, proteins, and nucleic acids from combinatorial libraries. PMID: 18265399 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818003 Mon, 01 Oct 2007 04:00:00 +0100 1818003 http://www.medworm.com/index.php?rid=1818002&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265400%26dopt%3DAbstract Authors: Blackshaw S, Croix BS, Polyak K, Kim JB, Cai L Serial analysis of gene expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point in the sequence of all cDNAs in the sample analyzed. This short tag, because of its presence in a defined point in the sequence, is typically sufficient to uniquely identify every transcript in the sample. SAGE allows one to generate a comprehensive profile of gene expression in any sample desired from as little as 100,000 cells or 1 microg of total RNA. SAGE generates absolute, rather than relative, measurements of RNA abundance levels, and this fact allows an investigator to readily and reliably compare data to those produced by other laboratories, making the SAGE data set increasingly useful as more data is... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818002 Mon, 01 Oct 2007 04:00:00 +0100 1818002 http://www.medworm.com/index.php?rid=1818001&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265401%26dopt%3DAbstract Authors: Antal C, Teletin M, Wendling O, Dgheem M, Auwerx J, Mark M In this unit, a procedure for post-mortem examination of mice and tissue collection is provided. This procedure is performed for post-mortem analysis of anatomical defects (necropsy) and histological analysis and/or tissue collection destined for molecular biology applications. In both cases, tissue preservation is the major issue, but the way to achieve it depends on the objective. When histological analysis is the aim, tissue preservation is achieved by rapid transfer into fixative solutions. In contrast, molecular biology applications require rapid freezing of tissue samples to preserve mRNA integrity. Consequently, performing both procedures simultaneously may be at the expense of the final product quality. PMI... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818001 Mon, 01 Oct 2007 04:00:00 +0100 1818001 http://www.medworm.com/index.php?rid=1818014&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265388%26dopt%3DAbstract Authors: This appendix provides selected properties of radioisotopes commonly used in the molecular biology laboratory. PMID: 18265388 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818014 Sun, 01 Jul 2007 04:00:00 +0100 1818014 http://www.medworm.com/index.php?rid=1818013&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265389%26dopt%3DAbstract Authors: Meisenhelder J, Bursik S The pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detecti... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818013 Sun, 01 Jul 2007 04:00:00 +0100 1818013 http://www.medworm.com/index.php?rid=1818011&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265391%26dopt%3DAbstract Authors: Thomason LC, Costantino N, Court DL This unit describes the procedure used to move portions of the E. coli genome from one genetic variant to another. Fragments of approximately 100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome. PMID: 18265391 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818011 Sun, 01 Jul 2007 04:00:00 +0100 1818011 http://www.medworm.com/index.php?rid=1818010&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265392%26dopt%3DAbstract Authors: Knoll JH, Lichter P, Bakdounes K, Eltoum IE Nonisotopic in situ hybridization can be used to determine the cellular location and relative levels of expression for specific transcripts within cells and tissues. RNA in specimen preparations is hybridized with a biotin- or digoxigenin-labeled probe, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described here, along with amplification of weak FISH signals. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here. PMID: 18265392 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818010 Sun, 01 Jul 2007 04:00:00 +0100 1818010 http://www.medworm.com/index.php?rid=1818006&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265396%26dopt%3DAbstract Authors: Ng P, Wei CL, Ruan Y The Paired-End diTagging (PET) procedure enables one to obtain sequence information from both termini of any contiguous DNA fragment. This is achieved by a series of enzymatic manipulations that introduce MmeI sites directly flanking each DNA insert during the construction of a plasmid library. Subsequent MmeI digestion and self-ligation results in the production of covalently-linked paired-end ditags (PETs) that can be extracted and then concatenated for efficient sequencing. By mapping the PET sequences to assembled genomes, the original DNA fragments from which the PETs were derived can be precisely localized. This unit details two applications of PET technology. In GIS-PET, ditagging of mRNA converted to full-length cDNA enables whole-transcriptome ana... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818006 Sun, 01 Jul 2007 04:00:00 +0100 1818006 http://www.medworm.com/index.php?rid=1818005&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265397%26dopt%3DAbstract Authors: Kim TH, Barrera LO, Ren B ChIP-chip combines chromatin immunoprecipitation (ChIP) with microarrays (chip) to determine protein-DNA interactions occurring in living cells. The high throughput nature of this method makes it an ideal approach for identifying transcription factor targets or chromatin modification sites along the genome. UNIT 21.9 describes a protocol for analysis of protein-DNA interactions in yeast cells. This unit introduces an alternative protocol developed for mammalian cells. PMID: 18265397 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818005 Sun, 01 Jul 2007 04:00:00 +0100 1818005 http://www.medworm.com/index.php?rid=1818012&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265390%26dopt%3DAbstract Authors: Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H, Datta S, Oppenheim A The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two-step method of making genetic alterations without leaving any unwanted changes, and a method for retr... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818012 Sun, 01 Apr 2007 04:00:00 +0100 1818012 http://www.medworm.com/index.php?rid=1818009&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265393%26dopt%3DAbstract Authors: Choi VW, Asokan A, Haberman RA, Samulski RJ Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection in many different cell types and its ability to persist and lead to long-term gene expression. The vector is also a valuable tool in molecular biology experiments. This unit describes efficient methods to generate high-titer, research-grade, adenovirus-free, recombinant single-stranded and self-complementary adeno-associated virus in various serotypes, along with methods to quantify the viral vectors. PMID: 18265393 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818009 Sun, 01 Apr 2007 04:00:00 +0100 1818009 http://www.medworm.com/index.php?rid=1818000&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265402%26dopt%3DAbstract Authors: Mark M, Teletin M, Antal C, Wendling O, Auwerx J, Heikkinen S, Khetchoumian K, Argmann CA, Dgheem M Due to the small size of the mouse, evaluating its clinical phenotype is sometimes problematic. In contrast, mouse models are readily accessible to post-mortem analyses at any time during the course of a disease and prior to its clinical onset. RNA, protein, and histological analyses following sacrifice represent a powerful means to identify affected cell types and molecular events underlying the altered phenotype, and therefore to understanding the signaling or metabolic pathways involved. In this unit, an overview of post-mortem analyses is provided with a strong emphasis on the principles of routine histology, including tissue fixation, processing, embedding, and staining wit... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818000 Sun, 01 Apr 2007 04:00:00 +0100 1818000 http://www.medworm.com/index.php?rid=1817998&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265404%26dopt%3DAbstract Authors: Elion EA, Marina P, Yu L This unit describes the use of PCR to construct hybrid DNA molecules. The unit provides an overview of how PCR can be exploited to accomplish numerous cloning strategies. The Basic Protocol outlines the PCR amplification and cloning strategies. The Commentary includes a troubleshooting guide for problems most frequently encountered in PCR cloning, plus four specific examples of the application of this technique to create in-frame fusion proteins, to create recombinant DNA products, to generate deletions and inversions by inverse PCR, and to introduce mutagenized PCR products or specific mutations or fusions by gap repair in yeast. PMID: 18265404 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817998 Sun, 01 Apr 2007 04:00:00 +0100 1817998 http://www.medworm.com/index.php?rid=1817997&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265405%26dopt%3DAbstract Authors: Greenberg ME, Bender TP Newly transcribed RNA can be identified using the nuclear runoff transcription assay. In this assay, isolated nuclei, free of membranes and cytoplasmic debris, are used in an in vitro transcription reaction in the presence of (32)P-labeled UTP. The labeled RNA can then be hybridized to cDNAs immobilized on nitrocellulose. This unit describes three methods for isolating suitable nuclei by detergent lysis, Dounce homogenization, and centrifugation on a sucrose gradient. Two Support Protocols describe the preparation of nitrocellulose filter strips containing double-stranded and single-stranded DNAs, which are used to detect the presence of specific transcripts in the nuclear runoff transcription assay. PMID: 18265405 [PubMed - indexed for MEDLINE] (So... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817997 Sun, 01 Apr 2007 04:00:00 +0100 1817997 http://www.medworm.com/index.php?rid=1818008&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265394%26dopt%3DAbstract Authors: Nühse T, Yu K, Salomon A The identification of protein phosphorylation sites from cell-derived proteins is crucial to the understanding of signal transduction pathways. While determining the modified sites on individual proteins can present a significant challenge, recent progress in the rapid, large-scale identification of phosphopeptides from complex protein mixtures by combinations of affinity chromatography and mass spectrometry provides a powerful tool to decipher the phosphoproteome. A set of protocols is described for sample preparation, fractionation, immobilized metal ion affinity chromatography (IMAC), and mass spectrometric analysis of phosphorylation sites. Parts of the protocols can be combined in different ways to adapt to sample amounts, complexity, and ava... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818008 Mon, 01 Jan 2007 05:00:00 +0100 1818008 http://www.medworm.com/index.php?rid=1818007&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265395%26dopt%3DAbstract Authors: Grant GR, Manduchi E, Stoeckert CJ Microarray experiments require careful planning and choice of analysis tools in order to get the most out of the data generated, especially considering the associated significant cost and effort. Microarray experiments also require careful documentation, often residing in local databases and/or submitted to public repositories. An often bewildering assortment of choices is available for experimental design, data preprocessing, data analysis (e.g., differential gene expression, classification), and data management. This unit covers the basic steps and common applications for planning, data processing, and data management of microarray experiments, and provides guidance to making choices based on the goals and practical realities of the experim... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818007 Mon, 01 Jan 2007 05:00:00 +0100 1818007 http://www.medworm.com/index.php?rid=1817999&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265403%26dopt%3DAbstract Authors: Heikkinen S, Argmann CA, Champy MF, Auwerx J Obesity and dyslipidemia are often found in association with insulin resistance (IR). These components combined with hypertension characterize the most common endocrine disorder in humans, the metabolic syndrome. Thus, in addition to profiling body weight evolution and lipid metabolites, glucose tolerance (a reflection of IR) and insulin sensitivity should also be considered as part of any metabolic phenotyping protocol. The ability to measure IR and glucose tolerance is important not only in the quest to fully understand the pathogenesis of the metabolic syndrome in the mouse, but also to test the effects of potential interventions. This unit presents a variety of tests used for this purpose, including direct blood glucose measurem... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1817999 Mon, 01 Jan 2007 05:00:00 +0100 1817999 http://www.medworm.com/index.php?rid=1818033&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265369%26dopt%3DAbstract Authors: Gallagher SR, Desjardins PR Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/l to 50 mg/ml. PMID: 18265369 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818033 Wed, 01 Nov 2006 05:00:00 +0100 1818033 http://www.medworm.com/index.php?rid=1818031&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265371%26dopt%3DAbstract Authors: Simonian MH, Smith JA This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradf... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818031 Wed, 01 Nov 2006 05:00:00 +0100 1818031 http://www.medworm.com/index.php?rid=1818024&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265378%26dopt%3DAbstract Authors: Elion EA Coprecipitation of proteins from whole-cell extracts is a valuable approach to test for physical interactions between proteins of interest. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The approaches described can be adapted for other systems. In a typical experiment, as described here, cells are lysed and a whole-cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid-phase affinity matrix, and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high affin... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818024 Wed, 01 Nov 2006 05:00:00 +0100 1818024 http://www.medworm.com/index.php?rid=1818015&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265387%26dopt%3DAbstract Authors: Porreca GJ, Shendure J, Church GM Polony DNA sequencing provides an inexpensive, accurate, high-throughput way to resequence genomes of interest by comparison to a reference genome. Mate-paired in vitro shotgun genomic libraries are produced and clonally amplified on microbeads by emulsion PCR. These serve as templates for sequencing by fluorescent nonamer ligation reactions on a microscope slide. Each sequencing run results in millions of 26-bp reads that can be aligned to the reference genome, allowing the identification of differences between sequences. PMID: 18265387 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818015 Wed, 01 Nov 2006 05:00:00 +0100 1818015 http://www.medworm.com/index.php?rid=1818029&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265373%26dopt%3DAbstract Authors: Gallagher SR Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodec... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818029 Tue, 01 Aug 2006 04:00:00 +0100 1818029 http://www.medworm.com/index.php?rid=1818028&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265374%26dopt%3DAbstract Authors: Abmayr SM, Yao T, Parmely T, Workman JL Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of th... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818028 Tue, 01 Aug 2006 04:00:00 +0100 1818028 http://www.medworm.com/index.php?rid=1818022&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265380%26dopt%3DAbstract Authors: Gilbert C, Svejstrup JQ Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818022 Tue, 01 Aug 2006 04:00:00 +0100 1818022 http://www.medworm.com/index.php?rid=1818019&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265383%26dopt%3DAbstract Authors: Argmann CA, Auwerx J There are many elements in plasma that can act as surrogate markers of the physiological well-being of a mouse, thus making the collection of blood and plasma a general technique with many applications in mouse phenotyping. For example, the presence of certain enzymes in plasma can serve as markers of tissue toxicity (AST, ALT) and general function, and the more sophisticated lipid and lipoprotein profile tests (cholesterol, LDL) can point to dyslipidemias. As many of the tests available to measure these parameters have been adapted to automated systems in a high-throughput fashion, they have become part of the first line of screening protocols in mouse phenotyping. In this section, general techniques associated with collection and processing of blood are ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818019 Tue, 01 Aug 2006 04:00:00 +0100 1818019 http://www.medworm.com/index.php?rid=1818017&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265385%26dopt%3DAbstract Authors: Argmann CA, Houten SM, Champy MF, Auwerx J Lipids are important body constituents that are vital for cellular, tissue, and whole-body homeostasis. Lipids serve as crucial membrane components, constitute the body's main energy reservoir, and are important signaling molecules. As a consequence of these pleiotropic functions, many common diseases, including atherosclerosis, chronic inflammatory disorders, and obesity, have been associated with altered lipid homeostasis. Lipid abnormalities are hence increasingly analyzed in mouse models. This unit describes commonly used methods to analyze mouse lipid metabolism, with techniques that evaluate lipids both in blood and in tissues. Despite the similarities between men and mice in many aspects of metabolism, important differences als... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818017 Tue, 01 Aug 2006 04:00:00 +0100 1818017 http://www.medworm.com/index.php?rid=1818032&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265370%26dopt%3DAbstract Authors: Phelan MC This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID: 18265370 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818032 Mon, 01 May 2006 04:00:00 +0100 1818032 http://www.medworm.com/index.php?rid=1818030&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265372%26dopt%3DAbstract Authors: Smith JA This unit describes a solution-phase radioimmunoassay (RIA) using the double antibody/PEG method. A solution RIA determines the amount of a specific protein (or peptide) in a sample by comparison to a standard curve of the same protein (or peptide). The amount of protein (or peptide) in the sample is determined based on its ability to compete with a radiolabeled tracer for binding to a specific antibody. A secondary antibody and PEG are used to precipitate the primary antibody-protein complexes. The amount of radioactivity in the complex is inversely proportional to the amount of protein in the sample. Under ideal conditions, an RIA is capable of measuring picograms of a protein (or peptide), but only if the tracer is labeled at a high specific activity. To this end, ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818030 Mon, 01 May 2006 04:00:00 +0100 1818030 http://www.medworm.com/index.php?rid=1818027&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265375%26dopt%3DAbstract Authors: Cruz F, Lee RT, Huang H Cellular morphology is inherently three-dimensional. However, most histological techniques for tissue analysis focus on extracting information from two-dimensional slices of fixed samples or dissociated cells. These techniques result in a significant loss of the three-dimensional information of the tissue, including true cell volume, orientation, and whole cell shape. This unit discusses various options for three-dimensional imaging, provides a protocol for performing post-processing reconstruction based on serial slicing, and discusses the current advantages and limitations of the three-dimensional approach to quantitative tissue analysis. The focus of this protocol is on cardiac tissue, but the techniques can be applied to any solid tissue. PMID: ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818027 Mon, 01 May 2006 04:00:00 +0100 1818027 http://www.medworm.com/index.php?rid=1818023&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265379%26dopt%3DAbstract Authors: Miele A, Gheldof N, Tabuchi TM, Dostie J, Dekker J Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells. ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818023 Mon, 01 May 2006 04:00:00 +0100 1818023 http://www.medworm.com/index.php?rid=1818016&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265386%26dopt%3DAbstract Authors: Park J, Labaer J The study of protein en masse, or functional proteomics, depends on the availability of full-length cDNAs in appropriate expression-ready plasmid vectors for protein expression and functional analysis. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence to be cloned, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using two of the most popular recombinational cloning technologies, now commercially available from Invitrogen (Gateway) and BD C... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818016 Mon, 01 May 2006 04:00:00 +0100 1818016 http://www.medworm.com/index.php?rid=1818026&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265376%26dopt%3DAbstract Authors: Bookout AL, Cummins CL, Mangelsdorf DJ, Pesola JM, Kramer MF Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818026 Wed, 01 Feb 2006 05:00:00 +0100 1818026 http://www.medworm.com/index.php?rid=1818025&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265377%26dopt%3DAbstract Authors: Shaul Y, Seger R Mitogen-activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The primary method describes the use of immunoblotting with specific anti-phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in-gel kinas... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818025 Wed, 01 Feb 2006 05:00:00 +0100 1818025 http://www.medworm.com/index.php?rid=1818021&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265381%26dopt%3DAbstract Authors: Argmann CA, Dierich A, Auwerx J As the focus of human genetics shifts from Mendelian traits to complex diseases, a sophisticated genetic tool kit-with space for genetics (classical, molecular, statistical, and quantitative), metabolics, proteomics, bioinformatics, and mathematics-is required to elucidate their multifactorial traits and regulatory processes. Importantly, mouse resources optimized to study the actions of isolated genetic loci on a fixed background are insufficient on their own for studying intact polygenic networks and genetic interactions, and researchers must work in the context of experimental model systems that optimally mimic the genetic structure of human populations. The success of such phenogenomic approaches depend on the efficacy by which specific muta... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818021 Wed, 01 Feb 2006 05:00:00 +0100 1818021 http://www.medworm.com/index.php?rid=1818020&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265382%26dopt%3DAbstract Authors: Argmann CA, Auwerx J Mouse models are increasingly popular tools to study and characterize the molecular and physiological bases for human diseases. Due to the readily available genetic tools to construct mouse models of complex diseases, the flow of efficient and vigilant mouse phenotyping has now become the bottleneck in mouse functional genomics. This unit addresses the importance of minimizing and defining confounding genetic and environmental sources of variability. PMID: 18265382 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818020 Wed, 01 Feb 2006 05:00:00 +0100 1818020 http://www.medworm.com/index.php?rid=1818018&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265384%26dopt%3DAbstract Authors: Argmann CA, Champy MF, Auwerx J Body mass and composition reflect the combined effects of three processes: energy intake, energy partitioning (storage), and energy expenditure. Energy is released from food as it is combusted to carbon dioxide and water, and is expended as heat and work within a cell. The energy stores, mainly in adipose tissue, represent the net balance between intake and expenditure. The methods outlined in this unit evaluate these three processes by measuring food intake and lipid absorption, body fat composition, and energy expenditure. Evaluation of food intake and fat mass is a useful first-line phenotyping test indicating altered energy homeostasis. Evaluation of energy expenditure in this unit addresses obligatory basal energy expenditure (for performan... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818018 Wed, 01 Feb 2006 05:00:00 +0100 1818018 http://www.medworm.com/index.php?rid=1818045&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265357%26dopt%3DAbstract Authors: Kim J, Iyer VR Sequence Tag Analysis of Genomic Enrichment (STAGE) is a method for experimentally identifying the in vivo chromosomal targets of DNA-binding proteins in any sequenced genome. STAGE generates 21-bp tags derived from DNA isolated by chromatin immunoprecipitation (ChIP; UNIT 21.3). Concatamers of tags are cloned and sequenced to yield a STAGE library. Tags in the library represent DNA fragments that were occupied by the DNA-binding protein, and mapping these tag sequences to the genome identifies the binding loci of the DNA-binding protein in vivo. STAGE can be applied to any sequenced genome to identify targets of DNA-binding proteins without requiring extensive microarray resources. PMID: 18265357 [PubMed - indexed for MEDLINE] (Source: Current Protocols in ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818045 Tue, 01 Nov 2005 05:00:00 +0100 1818045 http://www.medworm.com/index.php?rid=1818038&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265364%26dopt%3DAbstract Authors: Pfeffer S, Lagos-Quintana M, Tuschl T Small RNAs that are derived from dsRNA precursors act as guide RNAs during sequence-specific epigenetic regulation of eukaryotic gene expression. These small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into one or more of the following categories: small interfering RNAs (siRNAs), microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record the profile of small RNAs expressed in cultured cells or tissues are described. The small RNAs are directionally cloned after isolation from total RNA. The methods rely on T4 RNA ligase-based joining of adapter oligonucleotides to the 3' and 5' termini of the pool of small RNAs. The ligation products are reverse transcribed and PCR-amplified. It is recommended ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818038 Tue, 01 Nov 2005 05:00:00 +0100 1818038 http://www.medworm.com/index.php?rid=1818037&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265365%26dopt%3DAbstract Authors: Yin Y, Chory J, Baulcombe D RNA silencing in plants is a rapid and facile approach for assignment of gene function and virus resistance. It allows selective RNA degradation through a mechanism that is given specificity by short interfering RNAs. The RNA silencer sequences are normally delivered as transgenes or a part of virus-vectors. This unit provides Basic Protocols for transgene and virus induced silencing. It also includes a Basic Protocol for protein overexpression using viral proteins that suppress silencing. PMID: 18265365 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818037 Tue, 01 Nov 2005 05:00:00 +0100 1818037 http://www.medworm.com/index.php?rid=1818041&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265361%26dopt%3DAbstract Authors: Conner DA This unit describes strategies and methods used to establish a colony of transgenic mice. Basic mating and genotyping strategies are introduced. PMID: 18265361 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818041 Mon, 01 Aug 2005 04:00:00 +0100 1818041 http://www.medworm.com/index.php?rid=1818040&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265362%26dopt%3DAbstract Authors: Jeong Y, Epstein DJ Bacterial artificial chromosomes (BACs) are the vectors of choice for the construction of genomic DNA libraries and, as such, have proven instrumental in the generation of large-scale physical maps; positional cloning projects; and the sequencing of human, mouse, and a plethora of other genomes. A number of methods have recently been developed to modify BAC DNA (e.g., insertion, deletion, substitution), making BACs even more useful for functional genomic research. This unit describes two protocols for BAC modification in E. coli, one that allows for specific changes at a given DNA sequence and another that is more suited for rapid and nonspecific integration of foreign DNA (such as a reporter cassette) into a BAC insert. In addition, a simple and reliable m... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818040 Mon, 01 Aug 2005 04:00:00 +0100 1818040 http://www.medworm.com/index.php?rid=1818039&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265363%26dopt%3DAbstract Authors: Gulick J, Robbins J Transgenesis has proven useful in creating animal models that mimic certain disease states, providing a mechanistic approach for understanding the underlying disease mechanisms at the molecular and cellular levels. With traditional transgenics, the gene of interest is cloned behind a promoter that has the desired expression pattern, allowing the gene to be expressed in those tissues at the developmental times that the promoter is active. In order to more precisely control gene expression both in vitro and in vivo, inducible systems that use pharmacologic intervention to control transgene expression have been developed (UNIT 16.14). As previously described, the system consists of two components, an activator that is regulated by tetracycline and a responder ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818039 Mon, 01 Aug 2005 04:00:00 +0100 1818039 http://www.medworm.com/index.php?rid=1818034&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265368%26dopt%3DAbstract Authors: Villegas J, McPhaul M The establishment of skin fibroblast strains provides a vehicle by which biochemical and genetic studies may be applied. In addition to providing genetic material to study the basis of disease, fibroblast cell lines established from skin biopsies may provide the investigator with a model in which to study specific disease states or normal physiology. This unit provides methods for the biopsy, establishment, and culture maintenance of skin fibroblasts to be used in further scientific study. PMID: 18265368 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818034 Mon, 01 Aug 2005 04:00:00 +0100 1818034 http://www.medworm.com/index.php?rid=1818047&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265355%26dopt%3DAbstract Authors: Krishnamurthy N, Sjölander KV Prediction of molecular function of proteins has become an important task in the genomics era. A wide variety of sequence analysis tools are available to biologists for this task. We have selected one or two primary protocols for tasks such as domain detection, subcellular localization, and motif detection. We also present a strategy for integration of results from different protocols. All the resources needed for these protocols are accessible via publicly available Web servers and databases and require little or no computational expertise. PMID: 18265355 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818047 Sun, 01 May 2005 04:00:00 +0100 1818047 http://www.medworm.com/index.php?rid=1818043&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265359%26dopt%3DAbstract Authors: Geisberg JV, Struhl K Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (UNIT ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with t... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818043 Sun, 01 May 2005 04:00:00 +0100 1818043 http://www.medworm.com/index.php?rid=1818036&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265366%26dopt%3DAbstract Authors: Xu J Genetically manipulated transgenic and gene-targeted mouse models are indispensible tools used in defining the role of genes in development and organ function. Similarly, cells isolated from these mice bear the same genetic alterations and therefore can be extremely useful for studying the molecular and cellular mechanisms and regulatory gene networks involving the mutated gene under well defined culture conditions. In this unit, detailed protocols regarding isolation of mouse embryonic fibroblasts (MEFs) from mouse embryos, culture of MEFs, and immortalization of MEFs are described. These protocols should be particularly useful to those investigators who intend to establish primarily cultured MEFs and to immortalize primary MEFs into stable cell cultures with a permanent... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818036 Sun, 01 May 2005 04:00:00 +0100 1818036 http://www.medworm.com/index.php?rid=1818035&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265367%26dopt%3DAbstract Authors: Ling PD, Huls HM Recent advances in Genomics and Proteomics have necessitated a constant supply of DNA derived from specific genotypes. Lymphoblastoid cell lines (LCLs) are of great practical value as an unlimited source of stable genomic DNA and viable cells, which can be used to perform a variety of biochemical and molecular studies. LCLs are typically generated by infection of primary lymphocytes with Epstein-Barr virus (EBV). In this unit, we describe simple procedures for production of EBV, isolation of lymphocytes, EBV infection of lymphocytes, and isolation of the resulting LCLs. PMID: 18265367 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818035 Sun, 01 May 2005 04:00:00 +0100 1818035 http://www.medworm.com/index.php?rid=1818049&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265353%26dopt%3DAbstract Authors: Chakravarti B, Gallagher SR, Chakravarti DN One- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) have been widely used for the separation and quantitative estimation of proteins. Following electrophoresis, the gels are stained appropriately to visualize the proteins. Difference gel electrophoresis (DIGE) is a new technique in which different protein samples, individually labeled with specific CyDyes, are combined together followed by electrophoresis and post electrophoretic co-detection and co-analysis on the same gel. CyDye DIGE fluor minimal dyes, which consist of three different CyDyes with different spectral characteristics, have been widely used for such purposes. The technique is highly sensitive with a wide dynamic ran... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818049 Tue, 01 Feb 2005 05:00:00 +0100 1818049 http://www.medworm.com/index.php?rid=1818048&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265354%26dopt%3DAbstract Authors: Oka K, Chan L The helper-dependent adenovirus (HDAd) is a recently developed adenovirus-based vector with an improved safety profile and long-term transgene expression. In this unit, a basic procedure for HDAd production using the Cre-loxP system is presented. Amplification and large-scale production of the vector can be done in both adherent and suspension cell culture systems. Included are protocols for Southern blot analysis to monitor vector amplification, slot blot assay to determine the infectious titer of the purified HDAd, and real-time PCR to detect helper virus contamination in the preparation. PMID: 18265354 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818048 Tue, 01 Feb 2005 05:00:00 +0100 1818048 http://www.medworm.com/index.php?rid=1818046&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265356%26dopt%3DAbstract Authors: Zaret K This unit describes methodology for using micrococcal nuclease to investigate the presence of nucleosomes at a particular location in chromatin and to map the positions of nucleosomes at various levels of resolution. The approaches are readily adaptable to other probes of chromatin structure that cause DNA cleavage. Results obtained from such chromatin studies provide a structural view of the molecular environment of gene in their native context in cells. PMID: 18265356 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818046 Tue, 01 Feb 2005 05:00:00 +0100 1818046 http://www.medworm.com/index.php?rid=1818044&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265358%26dopt%3DAbstract Authors: Aparicio O, Geisberg JV, Sekinger E, Yang A, Moqtaderi Z, Struhl K Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein-DNA interactions at a pa... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818044 Tue, 01 Feb 2005 05:00:00 +0100 1818044 http://www.medworm.com/index.php?rid=1818042&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265360%26dopt%3DAbstract Authors: Katagiri F, Glazebrook J In expression profiling studies, it is often necessary to identify groups of genes with similar expression profiles in a variety of samples, and/or groups of samples with similar expression profiles. Each profile can be expressed as a single data point in a space with the same number of dimensions as there are parameters in the profiles. In this way, pattern discovery among expression profiles is translated into pattern discovery in the spatial distribution of data points. Hierarchical clustering is useful for clustering similarly behaving genes or samples at local levels and for displaying the results in a simple color-coded manner. K-means clustering can be used for discovery of well-defined clusters. Principal component analysis and self-organizing ... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818042 Tue, 01 Feb 2005 05:00:00 +0100 1818042 http://www.medworm.com/index.php?rid=1818057&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265344%26dopt%3DAbstract Authors: Xu D, Xu Y Protein databases have become a crucial part of modern biology. Huge amounts of data for protein structures, functions, and particularly sequences are being generated. Searching databases is often the first step in the study of a new protein. Comparison between proteins and between protein families in databases provides information about the relationship between proteins within a genome or across different species, and hence offers much more information than can be obtained by studying only an isolated protein. In addition, secondary databases derived from experimental databases are also widely available. These databases reorganize and annotate the data or provide predictions. The use of multiple databases often helps researchers understand the structure and functio... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818057 Mon, 01 Nov 2004 05:00:00 +0100 1818057 http://www.medworm.com/index.php?rid=1818056&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265345%26dopt%3DAbstract Authors: Smith A, Nelson RJ Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single-base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit. PMID: 1... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818056 Mon, 01 Nov 2004 05:00:00 +0100 1818056 http://www.medworm.com/index.php?rid=1818054&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265347%26dopt%3DAbstract Authors: Moqtaderi Z, Struhl K This unit describes the combination of chromatin immunoprecipitation (ChIP) with microarray hybridization to determine the genome-wide occupancy profile of a DNA-associated protein. After conventional ChIP, the immunoprecipitated material is amplified by a two-step process involving primer extension followed by PCR in the presence of a modified nucleotide. The amplified DNA is fluorescently labeled in a reaction that couples dye to the modified nucleotide, and the labeled sample is hybridized to a microarray representing a complete genome. This method allows the study of a protein's pattern of DNA association across an entire genome with no need for prior knowledge of potential DNA targets. PMID: 18265347 [PubMed - indexed for MEDLINE] (Source: Curren... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818054 Mon, 01 Nov 2004 05:00:00 +0100 1818054 http://www.medworm.com/index.php?rid=1818052&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265349%26dopt%3DAbstract Authors: Conner DA This unit describes methods for the production of transgenic mice by injection of DNA into zygotes, including fertilized-egg isolation, zygote injection, and oviduct reimplantation. Methods for the preparation of plasmid and BAC DNA suitable for microinjection are also presented. PMID: 18265349 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818052 Mon, 01 Nov 2004 05:00:00 +0100 1818052 http://www.medworm.com/index.php?rid=1818065&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265336%26dopt%3DAbstract Authors: Adams LD, Gallagher SR Two-dimensional gel electrophoresis is the combination of two high-resolution electrophoretic procedures (isoelectric focusing and SDS-polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first-dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS-slab gel and electrophoresed. The proteins in the first-dimension gel migrate into the second-dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. For very basic or very acidic proteins, two alternate protocols are p... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818065 Wed, 01 Sep 2004 04:00:00 +0100 1818065 http://www.medworm.com/index.php?rid=1818053&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265348%26dopt%3DAbstract Authors: Katagiri F, Glazebrook J Microarray technology allows simultaneous measurement of the mRNA levels of thousands of genes. This powerful technology has applications in addressing many biological questions that were not approachable previously; however, the enormous size of microarray data sets leads to issues of experimental design and statistical analysis that are unfamiliar to many molecular biologists. The type of array used, design of the biological experiment, number of experimental replicates, and statistical method for data analysis should all be chosen based on the scientific goals of the investigator. This overview presents a discussion of the relative merits and limitations of various methods with respect to some common applications of microarray experiments. PMID:... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818053 Wed, 01 Sep 2004 04:00:00 +0100 1818053 http://www.medworm.com/index.php?rid=1818050&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265351%26dopt%3DAbstract Authors: Brown T, Mackey K, Du T Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818050 Wed, 01 Sep 2004 04:00:00 +0100 1818050 http://www.medworm.com/index.php?rid=1818064&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265337%26dopt%3DAbstract Authors: Medberry S, Gallagher S, Moomaw B Gel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide-based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single-gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image-capture device, and the images can be easily manipulated using image analysis software. This... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818064 Sat, 01 May 2004 04:00:00 +0100 1818064 http://www.medworm.com/index.php?rid=1818063&cid=s_38087_67_f&fid=38087&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18265338%26dopt%3DAbstract Authors: Gallagher S, Winston SE, Fuller SA, Hurrell JG Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horsera... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818063 Sat, 01 May 2004 04:00:00 +0100 1818063