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Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 comm...
Source: Current Protocols in Molecular Biology - September 30, 2009 Category: Molecular Biology Authors: Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE Tags: Curr Protoc Mol Biol Source Type: journals

Heat-activatable primers for hot-start PCR and hot-start one-step RT-PCR: endpoint and real-time experiments.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Molecular Biology - September 30, 2009 Category: Molecular Biology Authors: Ashrafi EH, Paul N Tags: Curr Protoc Mol Biol Source Type: journals

The UCSC genome browser: what every molecular biologist should know.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 applicati...
Source: Current Protocols in Molecular Biology - September 30, 2009 Category: Molecular Biology Authors: Mangan ME, Williams JM, Kuhn RM, Lathe WC Tags: Curr Protoc Mol Biol Source Type: journals

Design, synthesis, and amplification of DNA pools for in vitro selection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - September 30, 2009 Category: Molecular Biology Authors: Hall B, Micheletti JM, Satya P, Ogle K, Pollard J, Ellington AD Tags: Curr Protoc Mol Biol Source Type: journals

In vitro selection of RNA aptamers to a protein target by filter immobilization.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - September 30, 2009 Category: Molecular Biology Authors: Hall B, Arshad S, Seo K, Bowman C, Corley M, Jhaveri SD, Ellington AD Tags: Curr Protoc Mol Biol Source Type: journals

Pathway Modulators and Inhibitors.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - June 30, 2009 Category: Molecular Biology Authors: Smith JA Tags: Curr Protoc Mol Biol Source Type: journals

Metabolic Radiolabeling of Animal Cell Glycoconjugates.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 t...
Source: Current Protocols in Molecular Biology - June 30, 2009 Category: Molecular Biology Authors: Diaz S, Varki A Tags: Curr Protoc Mol Biol Source Type: journals

Resources for Small Regulatory RNAs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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. P...
Source: Current Protocols in Molecular Biology - June 30, 2009 Category: Molecular Biology Authors: Bell GW, Lewitter F Tags: Curr Protoc Mol Biol Source Type: journals

Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - June 30, 2009 Category: Molecular Biology Authors: Davidson EA, Dlugosz PJ, Levy M, Ellington AD Tags: Curr Protoc Mol Biol Source Type: journals

Laser Microdissection-Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - June 30, 2009 Category: Molecular Biology Authors: Scanlon MJ, Ohtsu K, Timmermans MC, Schnable PS Tags: Curr Protoc Mol Biol Source Type: journals

Using PATIMDB to create bacterial transposon insertion mutant libraries.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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: 1...
Source: Current Protocols in Molecular Biology - April 1, 2009 Category: Molecular Biology Authors: Urbach JM, Wei T, Liberati N, Grenfell-Lee D, Villanueva J, Wu G, Ausubel FM Tags: Curr Protoc Mol Biol Source Type: journals

Two-hybrid dual bait system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 expressi...
Source: Current Protocols in Molecular Biology - April 1, 2009 Category: Molecular Biology Authors: Kotova E, Coleman T, Serebriiskii I Tags: Curr Protoc Mol Biol Source Type: journals

DNA cloning and engineering by uracil excision.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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. I...
Source: Current Protocols in Molecular Biology - April 1, 2009 Category: Molecular Biology Authors: Bitinaite J, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

Selection of transfected mammalian cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 wi...
Source: Current Protocols in Molecular Biology - April 1, 2009 Category: Molecular Biology Authors: Mortensen RM, Kingston RE Tags: Curr Protoc Mol Biol Source Type: journals

Selected suppliers of reagents and equipment.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - January 1, 2009 Category: Molecular Biology Tags: Curr Protoc Mol Biol Source Type: journals

Staining proteins in gels.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 f...
Source: Current Protocols in Molecular Biology - January 1, 2009 Category: Molecular Biology Authors: Sasse J, Gallagher SR Tags: Curr Protoc Mol Biol Source Type: journals

Overview of mRNA expression profiling using DNA microarrays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 prese...
Source: Current Protocols in Molecular Biology - January 1, 2009 Category: Molecular Biology Authors: Katagiri F, Glazebrook J Tags: Curr Protoc Mol Biol Source Type: journals

Pattern discovery in expression profiling data.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 clu...
Source: Current Protocols in Molecular Biology - January 1, 2009 Category: Molecular Biology Authors: Katagiri F, Glazebrook J Tags: Curr Protoc Mol Biol Source Type: journals

Recombineering-Based Procedure for Creating Cre/loxP Conditional Knockouts in the Mouse.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Molecular Biology - January 1, 2009 Category: Molecular Biology Authors: Bouvier J, Cheng JG Tags: Curr Protoc Mol Biol Source Type: journals

Detection of proteins on blot transfer membranes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 1, 2008 Category: Molecular Biology Authors: Sasse J, Gallagher SR Tags: Curr Protoc Mol Biol Source Type: journals

Using Cell-ID 1.4 with R for Microscope-Based Cytometry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 b...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Chernomoretz A, Bush A, Yu R, Gordon A, Colman-Lerner A Tags: Curr Protoc Mol Biol Source Type: journals

Visualization of microscopy-based spectral imaging data from multi-label tissue sections.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Mansfield JR, Hoyt C, Levenson RM Tags: Curr Protoc Mol Biol Source Type: journals

Dietary manipulation of mouse metabolism.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Feige JN, Lagouge M, Auwerx J Tags: Curr Protoc Mol Biol Source Type: journals

Ribonucleases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 pre...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Nichols NM, Yue D Tags: Curr Protoc Mol Biol Source Type: journals

RNA ligases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 oligoribonucle...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Nichols NM, Tabor S, McReynolds LA Tags: Curr Protoc Mol Biol Source Type: journals

DNA-dependent DNA polymerases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Kucera RB, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

Template-independent DNA polymerases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Yue D, Tabor S, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

RNA-dependent DNA polymerases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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' term...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Tzertzinis G, Tabor S, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

RNA polymerases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Paschal BM, McReynolds LA, Noren CJ, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

DNA repair enzymes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 su...
Source: Current Protocols in Molecular Biology - October 1, 2008 Category: Molecular Biology Authors: Evans TC, Nichols NM Tags: Curr Protoc Mol Biol Source Type: journals

Immunoblotting and immunodetection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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, ...
Source: Current Protocols in Molecular Biology - July 1, 2008 Category: Molecular Biology Authors: Gallagher S, Winston SE, Fuller SA, Hurrell JG Tags: Curr Protoc Mol Biol Source Type: journals

Production of monoclonal antibody supernatant and ascites fluid.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Molecular Biology - July 1, 2008 Category: Molecular Biology Authors: Yokoyama WM Tags: Curr Protoc Mol Biol Source Type: journals

Using Morpholinos to control gene expression.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 theref...
Source: Current Protocols in Molecular Biology - July 1, 2008 Category: Molecular Biology Authors: Moulton JD, Yan YL Tags: Curr Protoc Mol Biol Source Type: journals

Growth and manipulation of yeast.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Treco DA, Winston F Tags: Curr Protoc Mol Biol Source Type: journals

EMS and UV mutagenesis in yeast.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Winston F Tags: Curr Protoc Mol Biol Source Type: journals

Using CellProfiler for automatic identification and measurement of biological objects in images.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 p...
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Vokes MS, Carpenter AE Tags: Curr Protoc Mol Biol Source Type: journals

In situ polymerase chain reaction and hybridization to detect low-abundance nucleic acid targets.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Bagasra O Tags: Curr Protoc Mol Biol Source Type: journals

Interaction trap/two-hybrid system to identify interacting proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - ...
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R Tags: Curr Protoc Mol Biol Source Type: journals

Production of a heterozygous mutant cell line by homologous recombination (single knockout).Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Mortensen R Tags: Curr Protoc Mol Biol Source Type: journals

Production of a homozygous mutant embryonic stem cell line (double knockout).Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - April 1, 2008 Category: Molecular Biology Authors: Mortensen R Tags: Curr Protoc Mol Biol Source Type: journals

Chromosome conformation capture carbon copy technology.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Molecular Biology - October 1, 2007 Category: Molecular Biology Authors: Dostie J, Zhan Y, Dekker J Tags: Curr Protoc Mol Biol Source Type: journals

Overview of receptors from combinatorial nucleic acid and protein libraries.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - October 1, 2007 Category: Molecular Biology Authors: D Ellington A Tags: Curr Protoc Mol Biol Source Type: journals

Serial analysis of gene expression (SAGE): experimental method and data analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - October 1, 2007 Category: Molecular Biology Authors: Blackshaw S, Croix BS, Polyak K, Kim JB, Cai L Tags: Curr Protoc Mol Biol Source Type: journals

Tissue collection for systematic phenotyping in the mouse.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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,...
Source: Current Protocols in Molecular Biology - October 1, 2007 Category: Molecular Biology Authors: Antal C, Teletin M, Wendling O, Dgheem M, Auwerx J, Mark M Tags: Curr Protoc Mol Biol Source Type: journals

Radioactivity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Tags: Curr Protoc Mol Biol Source Type: journals

Safe use of radioisotopes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Authors: Meisenhelder J, Bursik S Tags: Curr Protoc Mol Biol Source Type: journals

E. coli genome manipulation by P1 transduction.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Authors: Thomason LC, Costantino N, Court DL Tags: Curr Protoc Mol Biol Source Type: journals

In situ hybridization and detection using nonisotopic probes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 [Pub...
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Authors: Knoll JH, Lichter P, Bakdounes K, Eltoum IE Tags: Curr Protoc Mol Biol Source Type: journals

Paired-end diTagging for transcriptome and genome analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Authors: Ng P, Wei CL, Ruan Y Tags: Curr Protoc Mol Biol Source Type: journals

ChIP-chip for genome-wide analysis of protein binding in mammalian cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Molecular Biology - July 1, 2007 Category: Molecular Biology Authors: Kim TH, Barrera LO, Ren B Tags: Curr Protoc Mol Biol Source Type: journals