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. Tue, 02 Mar 2010 17:32:29 +0100 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 http://www.medworm.com/index.php?rid=1818062&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%3D18265339%26dopt%3DAbstract Authors: Pizard A, Haramis A, Carrasco AE, Franco P, López S, Paganelli A Nonisotopic in situ hybridization using intact embryos or organs is an important method for determining the spatial distribution of RNAs. Because it allows the analysis of large numbers of samples, it is amenable to temporal expression studies and comparison between different genotypes. It offers sensitivity and reproducibility. In addition, histological details are not lost during the staining process. The protocols in this unit can be used for whole-mount in situ hybridization in Xenopus, mouse, and chicken embryos, as well as dissected organs from mouse and chicken. Preparation of digoxigenin-labeled riboprobes is also described. PMID: 18265339 [PubMed - indexed for MEDLINE] (Source: Current Protocols... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818062 Sat, 01 May 2004 04:00:00 +0100 1818062 http://www.medworm.com/index.php?rid=1818058&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%3D18265343%26dopt%3DAbstract Authors: Blethrow J, Zhang C, Shokat KM, Weiss EL Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild-type kinases. These engineered kinases, which are referred to as "analog-sensitive" or "-as" alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by -as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild-type kinase genes with engineered alleles, the in vivo activity of a -as kinase can be reversibly blocked with an allele-specific inhibitor. Thus, analog-sensitive kinases can be effective tools for d... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818058 Sat, 01 May 2004 04:00:00 +0100 1818058 http://www.medworm.com/index.php?rid=1818061&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%3D18265340%26dopt%3DAbstract Authors: Murphy CI, Piwnica-Worms H, Grünwald S, Romanow WG, Francis N, Fan HY This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild-type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector.... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818061 Sun, 01 Feb 2004 05:00:00 +0100 1818061 http://www.medworm.com/index.php?rid=1818060&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%3D18265341%26dopt%3DAbstract Authors: Murphy CI, Piwnica-Worms H, Grünwald S, Romanow WG, Francis N, Fan HY This unit describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production , how to maximize and scale up recombinant protein production, and how to purify recombinant proteins. PMID: 18265341 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818060 Sun, 01 Feb 2004 05:00:00 +0100 1818060 http://www.medworm.com/index.php?rid=1818059&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%3D18265342%26dopt%3DAbstract Authors: Murphy CI, Piwnica-Worms H, Grünwald S, Romanow WG, Francis N, Fan HY Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post-translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system. PMID: 18265342 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818059 Sun, 01 Feb 2004 05:00:00 +0100 1818059 http://www.medworm.com/index.php?rid=1818055&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%3D18265346%26dopt%3DAbstract Authors: Mendelsohn AR Protein-protein interactions play a critical role in biology. Disrupting a specific protein-protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two-hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified. PMID: 18265346 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818055 Sun, 01 Feb 2004 05:00:00 +0100 1818055 http://www.medworm.com/index.php?rid=1818051&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%3D18265350%26dopt%3DAbstract Authors: Worby CA, Dixon JE RNA interference (RNAi) can be used to silence genes in a number of species, including plants, nematodes, protozoans, Drosophila melanogaster, mouse embryos, and mammalian and Drosophila cell cultures. Drosophila cell culture provides the opportunity to study signal transduction pathways and protein function in a simple, well defined cell culture paradigm. Furthermore, because Drosophila are RNAi responsive, the results obtained from experiments performed on cultured cells can be confirmed in the whole organism. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce gene silencing in a highly specific manner. This phenomenon is efficacious and long lived, being passed to subsequent generations in Drosophila cell culture... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818051 Sun, 01 Feb 2004 05:00:00 +0100 1818051 http://www.medworm.com/index.php?rid=1818086&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%3D18265315%26dopt%3DAbstract Authors: Albright LM This unit presents tables of vectors described throughout the manual, organized by particular function. Individual entries in each table provide figure citations, some specific characteristics, and restriction site data for each vector, as well as sources when available. Restriction site information was generated using MAPSORT from sequence information that was compiled in part from published sequence data. PMID: 18265315 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818086 Sat, 01 Nov 2003 05:00:00 +0100 1818086 http://www.medworm.com/index.php?rid=1818084&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%3D18265317%26dopt%3DAbstract Authors: Sasse J, Gallagher SR In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). 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 alkali treatment that enhances subsequent staining of bound proteins. PMID: 18265317 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818084 Sat, 01 Nov 2003 05:00:00 +0100 1818084 http://www.medworm.com/index.php?rid=1818083&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%3D18265318%26dopt%3DAbstract Authors: Forsburg SL The fission yeast S. pombe provides an attractive alternative system to budding yeast S. cerevisiae for studies of fundamental cell biology. Fission yeast is a particularly powerful model for studies of cell cycle, chromosome dynamics, and polarity. Other areas of study are also expanding. Conceptually similar genetic tools are available in both systems, and both organisms have completely sequenced genomes. PMID: 18265318 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818083 Sat, 01 Nov 2003 05:00:00 +0100 1818083 http://www.medworm.com/index.php?rid=1818082&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%3D18265319%26dopt%3DAbstract Authors: Forsburg SL Fission yeast can be grown and maintained using similar culture methods to those employed for budding yeast. The optimal media for S. pombe is somewhat different than that employed for S. cerevisiae, although budding yeast media can be used if necessary. Good sterile technique is essential to prevent contamination by airborne molds or bacteria. PMID: 18265319 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818082 Sat, 01 Nov 2003 05:00:00 +0100 1818082 http://www.medworm.com/index.php?rid=1818081&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%3D18265320%26dopt%3DAbstract Authors: Forsburg SL This unit presents aspects specific to genetic and cytological manipulation of fission yeast, including mating type testing, crosses, preparingmaking diploids, and analysis of meiotic products, and basic methods of cell cycle synchronization and analysis. These methods are different from those used in budding yeast because they depend upon the distinct biology of S. pombe, particularly its unwillingness to be a diploid in normal growth conditions. Similarly, the different cell shape and growth behavior of S. pombe require different approaches to basic cell cycle analysis. PMID: 18265320 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818081 Sat, 01 Nov 2003 05:00:00 +0100 1818081 http://www.medworm.com/index.php?rid=1818080&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%3D18265321%26dopt%3DAbstract Authors: Forsburg SL Methods of transformation rely upon conditioning cells to take up DNA, and growing them under selective conditions to establish and maintain the plasmid or integration. Different methods may be used, including electroporation, treatment with lithium cations, or protoplast treatment which removes the cell wall. Protoplasts prepared as for transformation can also be induced to fuse with each other and undergo karyogamy, which provides a means of mating sterile strains. PMID: 18265321 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818080 Sat, 01 Nov 2003 05:00:00 +0100 1818080 http://www.medworm.com/index.php?rid=1818085&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%3D18265316%26dopt%3DAbstract Authors: Sasse J, Gallagher SR This unit describes protocols for detecting protein in a gel by either Coomassie blue staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. 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. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final suppo... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818085 Fri, 01 Aug 2003 04:00:00 +0100 1818085 http://www.medworm.com/index.php?rid=1818079&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%3D18265322%26dopt%3DAbstract Authors: Bearer EL Quantitative image analysis turns microscopic data that might otherwise be merely descriptive into reliable mechanistic information. This unit provides an overview of the types of analysis that have been useful in the past, as well as descriptions of emerging applications. In addition, two protocols are included for importing data and for the first steps of data manipulation in the more common analytical applications of the freeware, NIH Image and ImageJ, as well as commercially available imaging software, Adobe Photoshop. PMID: 18265322 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818079 Fri, 01 Aug 2003 04:00:00 +0100 1818079 http://www.medworm.com/index.php?rid=1818073&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%3D18265328%26dopt%3DAbstract Authors: Lee CS, Das R, Reed R In many methods currently used to analyze RNA-protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA-protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA-protein complexes. Moreover, the use of low-melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel. PMID: 18265328 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818073 Fri, 01 Aug 2003 04:00:00 +0100 1818073 http://www.medworm.com/index.php?rid=1818072&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%3D18265329%26dopt%3DAbstract Authors: Luo MJ, Reed R One of the major focuses of modern biology is to understand the dynamics of RNA-protein interactions, including factors that interact with mRNA, rRNA, tRNA, snRNA, hnRNA, siRNA, and viral RNA. Identification the direct interactions between proteins and RNA has greatly advanced our knowledge about the function of RNA-protein machines. UV crosslinking is a straightforward and powerful tool in this endeavor. PMID: 18265329 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818072 Fri, 01 Aug 2003 04:00:00 +0100 1818072 http://www.medworm.com/index.php?rid=1818071&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%3D18265330%26dopt%3DAbstract Authors: Zhou Z, Reed R Biological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA-protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity-chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA-hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron micr... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818071 Fri, 01 Aug 2003 04:00:00 +0100 1818071 http://www.medworm.com/index.php?rid=1818069&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%3D18265332%26dopt%3DAbstract Authors: Kingston RE, Chen CA, Rose JK This unit presents two methods of calcium phosphate-based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES-buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, b... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818069 Fri, 01 Aug 2003 04:00:00 +0100 1818069 http://www.medworm.com/index.php?rid=1818078&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%3D18265323%26dopt%3DAbstract Authors: Weiser DC, Shenolikar S Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible prote... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818078 Thu, 01 May 2003 04:00:00 +0100 1818078 http://www.medworm.com/index.php?rid=1818076&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%3D18265325%26dopt%3DAbstract Authors: Baulcombe D The history of RNA interference (RNAi) has unfolded rapidly since 1997 with a series of discoveries from plants, fungi, and animals. Initially, the interest was directed towards development of gene silencing technology that could be applied in research, medical therapy, and crop improvement. However, as the underlying mechanism was revealed, it became apparent that RNAi manifests a novel system of genetic regulation that was only hinted at by previous data. This overview unit gives a brief history of the field, describes the natural role of RNAi and related processes, identifies strategies for RNAi and cosuppression in plants and animals, and describes methods for detection and characterization of siRNA and miRNA. PMID: 18265325 [PubMed - indexed for MEDLINE] (... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818076 Thu, 01 May 2003 04:00:00 +0100 1818076 http://www.medworm.com/index.php?rid=1818075&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%3D18265326%26dopt%3DAbstract Authors: John M, Geick A, Hadwiger P, Vornlocher HP, Heidenreich O This unit provides information how to use short interfering RNA (siRNA) for sequence specific gene silencing in mammalian cells. Several ways for siRNA generation and optimisation, as well as recommendations for cell transfection are presented. PMID: 18265326 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818075 Thu, 01 May 2003 04:00:00 +0100 1818075 http://www.medworm.com/index.php?rid=1818074&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%3D18265327%26dopt%3DAbstract Authors: Conte D, Mello CC RNAi has become an essential tool in C. elegans research. Procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA and soaking in dsRNA solution are described in this unit. PMID: 18265327 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818074 Thu, 01 May 2003 04:00:00 +0100 1818074 http://www.medworm.com/index.php?rid=1818070&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%3D18265331%26dopt%3DAbstract Authors: Hollenbaugh D, Aruffo A, Jones B, Linsley P This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell-surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA. PMID: 18265331 [PubMed - indexed for MEDLINE] (Source: C... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818070 Thu, 01 May 2003 04:00:00 +0100 1818070 http://www.medworm.com/index.php?rid=1818068&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%3D18265333%26dopt%3DAbstract Authors: Waldman T, Lee C, Nishanian TG, Kim JS Human somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells. This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice. Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well-defined mutation in an endogenous gene. These modified cells can be studied both in vitro and in vivo. This unit presents protocols for human somatic cell gene targeting. PMID: 18265333 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818068 Thu, 01 May 2003 04:00:00 +0100 1818068 http://www.medworm.com/index.php?rid=1818067&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%3D18265334%26dopt%3DAbstract Authors: Potter H 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. In this unit, the describes the electroporation of mammalian cells and an outlines modifications for preparation and transfection of plant protoplasts. PMID: 18265334 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Molecular Biology) Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818067 Thu, 01 May 2003 04:00:00 +0100 1818067 http://www.medworm.com/index.php?rid=1818066&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%3D18265335%26dopt%3DAbstract Authors: Mortensen R, Chesnut JD, Hoeffler JP, Kingston RE Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 10(4) cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from t... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818066 Thu, 01 May 2003 04:00:00 +0100 1818066 http://www.medworm.com/index.php?rid=1818077&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%3D18265324%26dopt%3DAbstract Authors: Klein CA, Zohlnhöfer D, Petat-Dutter K, Wendler N The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi-quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are avai... Current Protocols in Molecular Biology journals http://www.medworm.com/rss/comments.php?id=1818077 Sat, 01 Feb 2003 05:00:00 +0100 1818077