bluuebunny/crossref_metadata_2025_split huggingface dataset:
Permeabilization Strategies to Study Protein Phosphorylation
Authors: Carter A. Nigel 
Published: June 2004

AbstractThis unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. These experiments generally utilize
[g‐32P]ATP as an exogenously added phosphate donor, although
[g‐32P]GTP can be used in specific cases. Procedures are outlined for performing a protein phosphorylation experiment using permeabilized cells and isolated intracellular organelles. Both of these procedures result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane‐bound kinases. Two different methods for determining the specific radioactivity of 32P ‐containing compounds are also included.
Phosphopeptide Mapping and Identification of Phosphorylation Sites
Authors: Meisenhelder Jill , Hunter Tony , van der Geer Peter 
Published: June 2004

AbstractThis unit describes a procedure for proteolytic digestion of a
32P‐labeled protein, followed by separation of the digestion products in two dimensions on a TLC plate, which gives rise to a phosphopeptide map. Phosphopeptides present on the TLC plate are visualized by autoradiography. These maps give information about the number of phosphate‐containing peptides in the digest, and this is related to the number of phosphorylation sites present in the protein. Phosphopeptide maps can also be used to find out whether the sites of phosphorylation on a protein change upon treatment of cells with certain agents. Procedures are also provided for the identification of the sites of phosphorylation from the phosphopeptide maps. A support protocol details purification of phosphopeptides by HPLC and subsequent analysis by mass spectrometry or peptide microsequencing.
Use of Protein Phosphatase Inhibitors
Authors: Weiser Douglas C. , Shenolikar Shirish 
Published: June 2004

AbstractReversible 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 protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting cellular phosphorylation activity with okadaic acid, microcystin‐LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.
Design and Use of Analog‐Sensitive Protein Kinases
Authors: Blethrow Justin , Zhang Chao , Shokat Kevan M. , Weiss Eric L. 
Published: June 2004

AbstractMany 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 discovery of the cellular functions and phosphorylation targets of individual enzymes. A theoretical background for the design and use of these alleles is discussed, as are strategies for construction of candidate –as alleles of any kinase.
Phage‐Based Expression Cloning to Identify Interacting Proteins
Authors: Stone Julie M. 
Published: June 2004

AbstractPhage‐based expression cloning is a simple, rapid, and powerful technique to identify interacting proteins. A protein of interest is expressed as a recombinant fusion protein and labeled with 32P at an engineered recognition site to facilitate detection. b‐gal proteins that are fused in‐frame to cDNA inserts in a phage‐derived expression library are produced by the phage and adsorbed onto nitrocellulose filters. The filters are then screened with the radioactive protein probe to identify phage clones that express the interacting protein. This technique leads directly to the isolation of a cDNA encoding the interacting protein, bypassing the need for labor‐intensive protein purification, microsequencing, or antibody production.
Detection of Protein‐Protein Interactions by Coprecipitation
Authors: Elion Elaine A. 
Published: June 2004

AbstractCoprecipitation 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 affinity to a molecule that can be coupled to a solid‐phase matrix.
Identification of Protein Interactions by Far Western Analysis
Authors: Edmondson Diane G. , Roth Sharon Y. 
Published: June 2004

AbstractFar western blotting is a method of identifying protein‐protein interactions. One protein of interest is immobilized on a solid support membrane, then probed with a non‐antibody protein. Far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways.
Micrococcal Nuclease Analysis of Chromatin Structure
Authors: Zaret Ken 
Published: June 2004

AbstractThis 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.
DNase I and Hydroxyl Radical Characterization of Chromatin Complexes
Authors: Vitolo Joseph M. , Thiriet Christophe , Hayes Jeffrey J. 
Published: June 2004

AbstractThe native chromatin complex within most eukaryotic nuclei is very difficult to study by biochemical means, so researchers have developed methods for studying smaller portions of the complex. This unit details the use of DNase I and hydroxyl radicals to characterize histone‐DNA interactions within such portions of the complex. DNase I digestion can be used to determine what regions of a DNA segment are intimately associated with the core histone proteins and what regions are more like naked DNA (i.e., linker DNA within the nucleosomal repeat). The finer deatils of histone‐DNA interactions and DNA structure within these complexes is best characterized by digestion with the hydroxyl radical. Both reagents may be used to assess the degree and homogeneity of rotational and translational positioning within isolated chromatin complexes.
Assembly of Nucleosomal Templates by Salt Dialysis
Authors: Lee Kyu‐Min , Narlikar Geeta 
Published: June 2004

AbstractBecause it is convenient to assemble nucleosomal templates through salt dialysis, large amounts of chromatin complexes can be made easily and in a short amount of time. This unit includes instructions for the various salt dialysis schemes (step versus gradient), which are accompanied by protocols for purification of core histones from bacteria and preparation of DNA for the nucleosomal arrays. Electrophoretic procedures to analyze the reconstituted complexes are also included.
Overview of Nucleic Acid Arrays
Authors: DeRisi Joseph 
Published: June 2004

AbstractNucleic acid array technology refers to the use and fabrication of arrays containing thousands of nucleic acid samples bound to solid substrates such as glass microscope slides or silicon wafers. Because the physical area occupied by each sample is usually 50 to 200 μm in diameter, it is possible to assay nucleic acid samples representing entire genomes, ranging in size from 3,000 to 32,000 genes, on a single slide. Microarrays are good for, among other things, analyzing gene expression patterns, genotyping and genetic mapping, comparative genomic hybridization, polysome analysis, and DNA‐protein interactions. This overview describes the technology and the uses, and provides valuable web site listings for readers to obtain additional information.
Preparation of mRNA for Expression Monitoring
Authors: Byrne Michael C. , Whitley Maryann Z. , Follettie Maximillian T. 
Published: June 2004

AbstractThe ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A+) vs. total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.
Mouse Embryonic Stem (ES) Cell Isolation
Authors: Conner David A. 
Published: June 2004

AbstractBuilding on the technology presented in earlier units of Chapter 23, this unit describes the basic method for the isolation of mouse embryonic (ES) stem cells. ES cells from wild‐type mice may be used for the production of mouse mutants by homologous recombination and blastocyst‐mediated transgenesis. ES cells from mutant mouse lines may be used in the analysis of mutant phenotypes.
Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)
Authors: Mortensen Richard 
Published: June 2004

AbstractHomozygous mutant embryonic stem (ES) cell lines have proven very useful in studying gene and protein function, and there are several reasons why this system might be preferred. Because many genes express their phenotype at the level of individual cells, using these cell lines eliminates the time and expense of producing a mutant animal. They can be isolated directly from a heterozygous mutant cell line without rederiving a cell line or using primary tissues from a mutant animal. In some cases, analysis of the phenotype of homozygous mutant cells can be performed more easily in culture. In addition, homozygous mutant ES cell lines may be produced even when a homozygous mutation is lethal to the animal. Finally, microinjection of homozygous mutant ES cells into blastocysts may allow investigation of the developmental potential of cells with such lethal mutations. This unit was moved from Chapter 9 to reflect a more appropriate placement, and has been updated and revised.
Chimeric Mouse Production by Microinjection
Authors: Conner David A. 
Published: June 2004

AbstractThe culmination of the creation of a mutation in mouse embryonic stem (ES) cells is, commonly, the generation of a mouse line that can propagate the mutation. The ability to combine methods of homologous recombination in ES cells with blastocyst‐mediated transgenesis has resulted in an explosion of tailored mutant mouse strains. These animals provide research tools that are virtually impossible to create using other methodologies. This unit describes the methods necessary to generate chimeras, from injection of the ES cells into the blastocoel cavity of 3.5‐day‐old embryos through the implantation of the injected embryos into the foster mother. The resultant pups are true chimeras: their tissues are derived from both the host embryo and from the ES cells. If the ES cells are able to populate the germ line, the chimera can pass an altered gene to offspring, resulting in a new mouse strain in which all cells contain an altered gene.
In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
Authors: Jhaveri Sulay D. , Ellington Andrew D. 
Published: June 2004

AbstractIn vitro selection of RNA aptamers that bind to a protein target is detailed in the protocols presented in this unit. Aptamers generated from these types of selection experiments can potentially function as protein inhibitors, and are often used as diagnostic or therapeutic reagents.
Peptide Aptamers: Dominant “Genetic” Agents for Forward and Reverse Analysis of Cellular Processes
Authors: Geyer C. Ronald 
Published: June 2004

AbstractPeptide aptamers are the newest in the class of “genetic” agents that aid in the analysis of cellular processes. Peptide aptamers interact with and can inactivate gene products, but don't mutate the DNA that encodes them. Reverse analysis with peptide aptamers involves isolating aptamers that interact with a specific protein and monitoring the resulting aptamer‐induced phenotype. Conversely, forward analysis with peptide aptamers involves expressing combinatorial libraries of aptamers within an organism and screening for aptamer‐induced variations in their phenotypes. The two‐hybrid system is used in both processes to help identify the protein interactions, and variations from the basic procedure as described in UNIT here are presented here.
Protein Selection Using mRNA Display
Authors: Keefe Anthony D. 
Published: June 2004

AbstractmRNA display is an in vitro technique that may be used to search natural or synthetic DNA libraries for the functional proteins and peptides they encode. mRNA‐displayed proteins are constructs in which a protein is covalently attached to the RNA that encodes it. This direct covalent association of phenotype (protein) and genotype (RNA) renders the protein directly amplifiable. This in turn allows successive cycles of selection, enrichment, and, optionally, mutagenesis, to be performed upon libraries of displayed proteins. At the end of this process, functional sequences will dominate the library; cloning and sequencing will reveal the identity of the selected functional proteins. mRNA display allows new functional proteins to be discovered without resorting to protein design. This unit describes generation of mRNA‐displayed proteins by the in vitro translation of mRNA display templates which are mRNA molecules 3'‐terminated in puromycin. Puromycin is a translation inhibitor that is able to enter the ribosome during translation and form a stable covalent bond with the nascent protein. This allows a stable covalent linkage to be formed between the mRNA display template and the protein it encodes, resulting in an mRNA‐displayed protein.
Laser Capture Microdissection
Authors: Frost Andra R. , Eltoum Isam‐Eldin , Siegal Gene P. 
Published: June 2004

AbstractLaser capture microdissection (LCM) offers a rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, or protein content, thereby allowing assessment of the role of the cell type in the normal physiologic or disease process being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LCM, including hematoxylin and eosin staining, are presented, as well as a protocol for the performance of LCM utilizing the PixCell I or II Laser Capture Microdissection System manufactured by Arcturus Engineering. Also provided is a protocol for tissue processing and paraffin embedding, and recipes for lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LCM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LCM.
Restriction‐Mediated Differential Display (RMDD)
Authors: Fischer Achim 
Published: June 2004

AbstractA variation on differential display, restriction‐mediated differential display (RMDD) presents an alternative approach to the fragment display technologies. It touts high sensitivity (detection of mRNAs at a dilution of 1:100,000 and of regulation factors lower than two‐fold), a strategy for avoiding false positives, nonradioactive detection, and universal applicability to any polyadenylated RNA.
Serial Analysis of Gene Expression (SAGE)
Authors: Blackshaw Seth , Kim Jae B. , Croix Brad St. , Polyak Kornelia 
Published: June 2004

AbstractSerial 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 defined presence in the sequence, is typically sufficient to uniquely identify every transcript in the sample. A comprehensive profile of gene expression in any sample can thus be generated. Absolute, rather than relative, measurements of RNA abundance levels are made, which will be useful in the reliable comparison of data between laboratories.Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point
Representational Difference Analysis
Authors: Chang Yuan 
Published: June 2004

AbstractRepresentational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detection of differences between two complex genomes. In this unit, protocols start with the restriction digestion of two comparison DNA samples. Specific linkers are ligated to fragments from each pool and amplicons are generated by PCR. Linkers are removed from both samples and a new linker is added only to size‐selected tester amplicons. These tester amplicons are mixed with a large excess of driver amplicons lacking linkers. Hybridization results in three species of dsDNA fragments: (1) both strands derived from driver DNA (lacking linkers on either strand), (2) hybrids with one strand from driver (no linker) and one from tester (with linker), and (3) both strands from tester DNA (linkers on both strands). Excess driver removes DNA fragments common to both samples, and only the DNA fragments unique to the tester are amplified with linker‐specific primers.Representational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detect Representational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detect
Safe Use of Hazardous Chemicals
Authors: Lunn George , Lawler Gretchen 
Published: June 2004

AbstractThis appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R. 
Published: June 2004

AbstractReliable quantitation of microgram, nanogram, and even picogram amounts of DNA and RNA in solution is essential to researchers using molecular biology techniques. In addition to the traditional absorbance measurements at 260 nm, three more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 25 pg DNA/ml to 50 mg DNA/ml.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractThe first section of this unit discusses basic principles of sterile technique, while the second section discusses medium preparation. The describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An describes freezing suspension cells.
Animal Health Assurance
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractA program to assure the health of laboratory animals and to provide a stable environment for their maintenance is necessary to reduce unwanted variables or complicating factors in experimentation. To assure animal health throughout a research study, the animals involved should be procured in good health, monitored for continued health status, and protected from pathogenic organisms through testing of any biological materials. This unit addresses all three of these health management practices. Essential in support of these practices is a diagnostic laboratory capable of testing for rodent and rabbit pathogens.
Isolation of Murine Natural Killer Cells
Authors: Reichlin Amy , Iizuka Koho , Yokoyama Wayne M. 
Published: June 2004

AbstractThis unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by cytotoxic depletion of contaminating cells with selected monoclonal antibodies (MAbs), complement lysis, and density‐gradient centrifugation to eliminate dead cells. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, are not at risk of activation by antibody cross‐linking. Purity can then be assessed by cell surface phenotype.
Production of Antibodies that Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Stern David F. , Roussel Robert R. 
Published: June 2004

AbstractIt is possible to produce anti‐phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross‐react with either the cognate unphosphorylated protein or other phosphoproteins. This unit describes production of antibodies against tyrosine‐phosphorylated peptides, with which the authors have the most expertise, but the principles discussed here also apply to peptides phosphorylated on serine and threonine. Protocols are presented for the production of polyclonal and monoclonal anti‐phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to an affinity matrix (Affi‐Gel 10); BSA‐agarose affinity matrix is commercially available.