bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of Protein Extracts from Yeast
Authors: Dunn Barbara , Wobbe C. Richard
Published: June 2004

Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial‐strength blender in the presence of liquid nitrogen.
Overview of Schizosaccharomyces pombe
Authors: Forsburg Susan L.
Published: June 2004

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.
Growth and Manipulation of S. pombe
Authors: Forsburg Susan L.
Published: June 2004

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.
Introduction of DNA into S. pombe Cells
Authors: Forsburg Susan L.
Published: June 2004

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.
In Situ Hybridization to CellularRNA
Authors: Zeller Rolf , Rogers Melissa , Haramis Anna G. , Carrasceo André s E.
Published: June 2004

In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide‐mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S‐labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin‐labeled RNA probes, which are non‐radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.
Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides
Authors: Zeller Rolf , Rogers Melissa
Published: June 2004

The morphology of specimen sections and the identity of specific areas are defined by lightly counterstaining sections. After staining, slides are dehydrated, mounted with coverslips, hardened, and cleaned for examination under the microscope. In the protocols provided in this unit, Giemsa stains predominantly the nuclei, hematoxylin/eosin stain differentiates both the nuclei and cytoplasm, toluidine blue staining is a simpler procedure that lightly stains both nuclei and cytoplasm, and Hoechst staining of nuclei provides a fast, easy, and effective way to simultaneously view the entire tissue and the regions of hybridization.
Immunohistochemistry
Authors: Watkins Simon
Published: June 2004

This unit surveys immunohistochemical techniques for protein localization. The first four protocols focus on indirect immunofluorescence which is the easiest and most widely used method of optical detection. Additional protocols present methods suitable for bright‐field microscopy, such as immunoperoxidase and immunogold labeling. The commentary contains an extensive troubleshooting guide with a list of suitable routine controls. The chosen method of tissue or cellular preparation must be suitable to answer the fundamental objective of the protocol (i.e., where the protein is localized within the system). Furthermore, the choice of fluorochrome and the method of labeling are also important to optimize the sensitivity of antigen localization. The basic protocol is therefore divided into two sections: (1) cellular preparations (monolayers of adherent cells and cells grown in suspension) and (2) tissue section labeling.
Whole‐Mount In Situ Hybridization and Detection ofRNAs in Vertebrate Embryos and Isolated Organs
Authors: Pizard Anne , Haramis Anna , Carrasco Andrés E. , Franco Paula , López Silvia , Paganelli Alejandra
Published: June 2004

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 inXenopus, mouse, and chicken embryos, as well as dissected organs from mouse and chicken. Preparation of digoxigenin‐labeled riboprobes is also described.
Basic Confocal Microscopy
Authors: Smith Carolyn L.
Published: June 2004

Confocal microscopy produces sharp images of structures within relatively thick specimens (up to several hundred microns). It is particularly useful for examining fluorescent specimens, such as fluorescent‐stained cells or intact organisms (e.g., Drosophila or zebrafish embryos), and can localize intracellular antigens in dissociated cells. Its sensitivity even allows the monitoring of fluorescence in living organisms, making it possible to follow the movements of fluorescent probes such as GFP or dyes for specific organelles. This unit presents the theory of optical sectioning, describes the instrumentation of the technique, and offers practical guidelines for sample preparation and analysis.
Direct DNA Sequencing of PCR Products
Authors: Dorit Robert L. , Ohara Osamu , Hwang Charles B‐C. , Kim Jae Bum , Blackshaw Seth
Published: June 2004

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam‐Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by “asymmetric PCR,” where one primer is present in vast excess over the other. This single‐stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end‐labeled products are also presented.
Enzymatic Amplification of RNA by PCR (RT‐PCR)
Authors: Beverly Stephen M.
Published: June 2004

Methods for enzymatic amplification of RNA by the polymerase chain reaction (RT‐PCR) are highlighted in this unit. The is especially useful for rare RNAs because all steps (annealing, reverse transcription, and amplification) are performed under optimal conditions, thereby maximizing efficiency and recovery. These are also important considerations when amplifying heterogeneous RNA populations or large RNAs. Although there are many commercially available kits for this procedure, the protocols listed here are good solid protocols that will work and that have withstood the test of time.
cDNA Amplification Using One‐Sided (Anchored) PCR
Authors: Dorit Robert L. , Ohara Osamu
Published: June 2004

The polymerase chain reaction (PCR) offers an opportunity to directly select, amplify, and isolate a message of interest. This unit describes anchored PCR, a modification of the standard reaction that allows amplification of full‐length mRNA when only a small amount of sequence information is available. Anchored PCR can be employed when only a small region of sequence lying within the mRNA is known in advance, unlike the amplification of RNA or cDNA by conventional PCR which requires prior knowledge of the sequences flanking the region of interest to design the PCR primers.
Overview of Protein Expression in E. coli
Authors: Schendel Paul F.
Published: June 2004

This overview unit introduces general considerations and strategies for expressing proteinsin E. coli. E. coli has two characteristics that make it ideally suited as an expression system for many kinds of proteins: it is easy to manipulate and it grows quickly in inexpensive media. These characteristics, coupled with more than 10 years' experience with expression of foreign genes, have established E. coli as the leading host organism for most scientific applications of protein expression. Despite a growing literature describing successful protein expression from cloned genes, each new gene still presents its own unique expression problems. There is no single set of methods that can guarantee successful production of every protein in a useful form. Nevertheless, the vast body of accumulated knowledge has led to a general approach that often helps to solve specific expression problems.
Expression Using the T7 RNA Polymerase/Promoter System
Authors: Tabor Stanley
Published: June 2004

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (pT7). In the , two plasmids are maintained within the same E. coli cell. One (the expression vector) contains pT7 upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat‐inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of pT7. If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.
Expression Using Vectors with Phage λ Regulatory Sequences
Authors: Shatzman Allan R. , Gross Mitchell S. , Rosenberg Martin
Published: June 2004

In the expression system described here, plasmids (pSKF) utilize regulatory signals‐such as the powerful promoter pL ‐from the bacteriophage λ. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the λ repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the λ genome. This so‐called defective lysogen supplies the λ regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL‐directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage λ antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome‐recognition and translation‐initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see first and second basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of “authentic“ gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.
Expression and Purification of Glutathione‐S‐Transferase Fusion Proteins
Authors: Smith Donald B. , Corcoran Lynn M.
Published: June 2004

This unit describes how pGEX vectors can be used in bacterial systems to express foreign polypeptides as fusions with glutathione‐S‐transferase (GST). In general, such fusion proteins are soluble and are easily purified from lysed cells under nondenaturing conditions by absorption with glutathione‐agarose beads, followed by elution in the presence of free glutathione. Potential applications of the pGEX vectors include the expression and purification of individual polypeptides (including short peptides) for use as immunogens and as biochemical and biological reagents, and in the construction of cDNA expression libraries. This protocol describes production and screening of pGEX transformants and purification of milligram quantities of fusion proteins from 1‐liter cultures. The commentary describes several modifications to the expression and purification protocol that may be useful in cases where fusion proteins are insoluble or unstable.
Expression of Proteins Using Semliki Forest Virus Vectors
Authors: Liljeström Peter , Garoff Henrik
Published: June 2004

Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein‐encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein‐coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self‐encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome‐mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
Special Considerations for Glycoproteins and Their Purification
Authors: Freeze Hudson H.
Published: June 2004

This unit begins by describing some properties of glycoproteins (e.g., subcellular location and solubility) that may be useful in determining which purification techniques to try. This discussion is followed by two protocols describing preparative glycoprotein purification using lectin‐affinity chromatography, as well as an outline for a small‐scale pilot procedure designed to check lectin binding and elution conditions. Lectins are often used for purifying glycoproteins because, in contrast to conventional purification procedures (e.g., gel filtration and ion‐exchange chromatography) that exploit general physical properties of glycoproteins, lectins recognize specific three‐dimensional structures created by a cluster of sugar residues. Conventional purification procedures are generally tried before applying lectin‐affinity chromatography.
Special Considerations for Glycolipids and Their Purification
Authors: Smith David F. , Prieto Pedro A.
Published: June 2004

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.
Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine
Authors: Zachara Natasha E. , Hart Gerald W. , Cole Robert N. , Gao Yuan
Published: June 2004

The modification of Ser and Thr residues with O‐linked b‐N‐acetyl glucosamine (O‐GlcNAc) is a common and essential modification of nuclear and cytoplasmic proteins, and it is thought that O‐GlcNAc performs a regulatory role in the cell. This unit concentrates on the techniques for the detection and analysis of proteins modified by O‐GlcNAc as well as methods for the analysis of enzymes responsible for the addition and removal of this group.
Lectin Analysis of Proteins Blotted onto Filters
Authors: Freeze Hudson H.
Published: June 2004

Lectins are proteins that bind with great specificity to certain carbohydrate structures. Plant lectins are widely used for investigations of carbohydrate structure and for fractionation and purification of individual oligosaccharides and glycopeptides. This unit describes the use of lectins as sensitive indicators for the presence of certain carbohydrate structures linked to proteins blotted onto filters. A tagged lectin is incubated with a blot containing the target protein and binding of the lectin is detected by one of several different procedures. Direct approaches include using lectins labeled with 125I or conjugated to horseradish peroxidase or alkaline phosphatase, which can be detected by chromogenic or luminescent visualization systems. Indirect approaches involve using lectins conjugated to biotin or digoxigenin followed by a second incubation with alkaline phosphatase‐conjugated avidin or antibodies specific for the haptenic digoxigenin group and then by visualization. Several commercial kits are available that provide labeled lectins, control proteins, and developing reagents needed for visualization. These systems can also be adapted for use with lectins other than those supplied with kits. The protocol in this unit is easy to perform with or without a kit. However, the results, while suggestive of carbohydrate structure, are not definitive.
Detection of Glycophospholipid Anchors on Proteins
Authors: Doering Tamara L. , Englund Paul T. , Hart Gerald W.
Published: June 2004

Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI‐anchored proteins. First, the detergent‐partitioning behavior of a protein of interest is examined for characteristics of GPI‐linked species. The protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.
Inhibition of Glycolipid Biosynthesis
Authors: Manzi Adriana E.
Published: June 2004

Adequate inhibition of glycolipid biosynthesis allows the study of their biological functions. The method presented in this unit employs a synthetic analog of ceramide, PDMP (1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol), that inhibits glycolipid biosynthesis in cultured cells. Optimum conditions for inhibition of glycolipid biosynthesis are determined, glycolipids extracted from cultures grown with and without inhibitor, and the patterns of glycolipids analyzed by HPTLC. Detection is achieved using colorimetric reactions, or by monitoring radioactivity when cells have been metabolically radiolabeled.
β‐Elimination for Release of O‐Linked Glycosaminoglycans from Proteoglycans
Authors: Conrad H. Edward
Published: June 2004

O‐linked glycosaminoglycan (GAG) chains in proteoglycans are readily released from their core proteins by treatment with alkali at room temperature. This β‐elimination is the same type of reaction as that for releasing O‐linked oligosaccharides from their core proteins. Under the reaction conditions described here, N‐linked oligosaccharides remain attached to the core protein, but any O‐linked oligosaccharides will be released along with the GAG chains. The procedure can be used to isolate the free GAG chains, the free O‐linked oligosaccharides, and the core protein (which will still have any N‐linked oligosaccharides that were originally present).
Analysis of Monosaccharides
Authors: Manzi Adriana E. , Powell Leland D. , Varki Ajit
Published: June 2004

This unit presents methods for assaying sialic acids, reducing sugars, and hexosamines. The BCA assay detects free reducing terminii in sugars released from glycoconjugates by appropriate treatments. Assays employing Ehrlich reagent (DMAB) detect hexosamines and N‐acetylhexosamines, including a method for hydrolyzing the glycosidic linkages of the hexosamines and a method for re‐N‐acetylation. The TBA and DMB assays can be used to quantitate and fractionate free forms of many types of sialic acids. Techniques for liberating the sialic acids from the parent glycoconjugates are also provided.
Analysis of Oligosaccharide Negative Charge by Anion‐Exchange Chromatography
Authors: Varki Ajit
Published: June 2004

This unit presents the analysis of negative charge on labeled N‐ or O‐linked oligosaccharides. These protocols may be used in the initial screening of oligosaccharides to detect negative charge, for analytical or preparative separation of oligosaccharides based on their negative charge, or to analyze the type of negative charge found on the oligos accharides. The describes the use of anion‐exchange (QAE‐Sephadex) chromatography with stepwise elution for estimating the number of negative charges on an oligosaccharide sample derived from glycosidase treatment of a glycoprotein. In an , gradient elution is used for the preparative separation of oligosaccharides based on negative charge. A describes a method for measuring loss of or change in negative charge after treatment of the oligosaccharide sample with mild acid and/or phosphatases.
HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides
Authors: Manzi Adriana E. , Hayes Bradley K.
Published: June 2004

This unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion‐exchange high‐performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge‐to‐mass ratio can also contribute to elution position.
Overview of Protein Phosphorylation
Authors: Sefton Bartholomew M. , Shenolikar Shirish
Published: June 2004

This overview discusses the significance and roles of protein phosphorylation in regulation of protein function. Sites of phosphorylation are described as well as methods for detecting both radiolabeled and unlabeled phosphoamino acids. Importantly, protein kinases and phosphatases, the regulators of phosphorylation are discussed.
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Authors: Sefton Bartholomew M.
Published: June 2004

This unit describes 32Pi labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The approach is appropriate, however, for labeling any cellular constituent with 32P. This procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The general approach involves biosynthetic labeling with 32Pi in medium containing a reduced concentration of phosphate. This approach can also be modified to label any cellular constituents with 32Pi. The first procedure described is 32Pi labeling of adherent or nonadherent (e.g., hematopoietic) cells with subsequent lysis in a detergent buffer. More rigorous lysis conditions to be used for working with proteins that are difficult to solubilize are also described.
Phosphoamino Acid Analysis
Authors: Sefton Bartholomew M.
Published: June 2004

It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial acid hydrolysis in HCl followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.
Detection of Phosphorylation by Enzymatic Techniques
Authors: Shenolikar Shirish
Published: June 2004

Reversible protein phosphorylation is an important mechanism for regulating physiological processes in both plant and animal cells. There are a number of techniques to demonstrate the presence of covalently bound phosphate in proteins. The general strategy of the protocols in this unit is to first examine the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Protein phosphatases that selectively hydrolyze phosphoserine and phosphothreonine or phosphotyrosine residues can then be used to identify a functionally important covalent modification. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as 32Pi based on its ability to form a complex with ammonium molybdate.
Assays of Protein Kinases Using Exogenous Substrates
Authors: Carter A. Nigel
Published: June 2004

In studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities: cyclic nucleotide‐dependent kinases, protein kinase C and isoforms, casein kinases, Ca2+/calmodulin‐dependent kinases, and tyrosine kinase. A protocol for in‐gel assays for specific kinase activity is also provided. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases.
Sequence Databases: Integrated Information Retrieval and Data Submission
Authors: Weisemann Jane M. , Boguski Mark S. , Ouellette B.F. Francis
Published: June 2004

This unit provides an overview of biomedical information resources, focusing on sequence data, structure information, and the associated literature, and also discusses how nucleotide sequence data gets into the databases in the first place. Some specific databases covered here are MEDLINE, GenBank, and Entrez.
Protein Databases on the Internet
Authors: Xu Dong , Xu Ying
Published: June 2004

This unit provides a starting point for readers to explore the potential of protein databases on the Internet. Databases for different aspects of proteins are discussed, with the focus on sequence, structure, and family. The strengths and weaknesses of the databases are addressed. From hundreds of on‐line protein databases, several major databases are discussed as examples to illustrate their features and how they can be used effectively. Most other protein databases can be explored in a similar way.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Finley Russell L. , Kolonin (hunt by interaction mating) Mikhail G. , Gyuris Jeno , Brent Roger
Published: June 2004

The parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two‐hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.
Surface Plasmon Resonance for Measurements of Biological Interest
Authors: Bamdad Cynthia
Published: June 2004

Genetic manipulations, including gene knock‐outs and mutant screens, provide an initial hint as to the function of a gene product and indicate possible associated factors. To unravel complicated biological processes, which control the development of organisms, one must identify the interacting components. An in vitro technique based on an optical phenomenon, called surface plasmon resonance (SPR), can simultaneously detect interactions between unmodified proteins and directly measure kinetic parameters of the interaction. This technique is gaining popularity, due to the increased availabilty oif user‐friendly machines, and an overview of the technology is presented in this unit.
Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant Proteins
Authors: Mendelsohn Andrew R.
Published: June 2004

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.
Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo
Authors: Aparicio Oscar , Geisberg Joseph V. , Struhl Kevin
Published: June 2004

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 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‐link 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 particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome‐wide basis. This unit describes the ChIP protocol for Saccharomyces cerevisiae; however, it is also applicable to other organisms.
Isolation of Histones and Nucleosome Cores from Mammalian Cells
Authors: Schnitzler Gavin R.
Published: June 2004

In vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left‐handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone‐containing particle is properly termed the nucleosome (or chromatosome), while the linker histone‐free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono‐ and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.
Profiling Human Gene Expression with cDNA Microarrays
Authors: Jiang Yuan , Lueders John , Glatfelter Arthur , Gooden Chris , Bittner Michael
Published: June 2004

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3' ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.
Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation
Authors: Conner David A.
Published: June 2004

Mitotically inactive mouse embryo fibroblasts (MEFs) are commonly used as feeder layers to prevent the differentiation of mouse embryonic stem (ES) cells. This unit describes the isolation of MEFs and the use of g‐irradiation or mitomycin C to produce inactivated feeders suitable for the culture of ES cells.
Mouse Embryonic Stem (ES) Cell Culture
Authors: Conner David A.
Published: June 2004

Mouse embryonic stem (ES) cells are used to generate mouse mutants by gene targeting and blastocyst‐mediated transgenesis. ES cells must be cultured under conditions that prevent differentiation to maintain their ability to transmit altered alleles by contributing to the germ line. This unit describes one of the most common methods for culturing ES cells using mouse embryo fibroblast (MEF) feeder layers and recombinant leukemia inhibitory factor (LIF) to prevent differentiation.
Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
Authors: Mortensen Richard
Published: June 2004

Formerly UNIT 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV‐TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes.
Mouse Colony Management
Authors: Conner David A.
Published: June 2004

The management of mouse colonies created by gene targeting is described in this unit. It defines guidelines necessary to establish the colony, and describes screening for germline transmission, marking the mice for easy and consistent identification, and genotyping DNA by PCR or Southern blotting analysis. It also reviews the software currently available for colony data management.
Design, Synthesis, and Amplification of DNA Pools for Construction of Combinatorial Pools and Libraries
Authors: Pollard Jack , Bell Sabine D. , Ellington Andrew D.
Published: June 2004

This unit describes the design, synthesis, and amplification of random‐sequence DNA pools, from which functional nucleic acid‐binding or catalytic species can be selected. Since it is an expensive and time‐consuming process, the authors have provided an extensive strategic planning section to guide investigators in designing and constructing the pool.
Preparation of Single Cells from Solid Tissues for Analysis by PCR
Authors: Sawtell N.M.
Published: June 2004

This unit details a protocol for the separation of solid tissues into single cell suspensions for subsequent analysis of nucleic acids and protein.Tissues are fixed in situ by perfusion, which terminates cell processes and thus changes that would accompany dissociating the living tissue. The procedure is particularly useful when the cell type of interest represents a minor population relative to other cells types in the tissue. Once separated, individual cells or groups of a particular cell type can then be analyzed using PCR strategies. The procedure can also be adapted to allow the quantification of the number of cells within a tissue containing specific nucleic acid sequences, for example, a particular viral DNA or RNA sequence.
PCR‐Based Subtractive cDNA Cloning
Authors: Patel Mukesh , Sive Hazel
Published: June 2004

Subtractive cloning is a powerful technique that allows isolation and cloning of mRNAs differentially expressed in two cell populations. In the generalized subtraction scheme the cell types to be compared are the [+] or tracer cells and the [‐] or driver cells, where mRNAs expressed in the tracer and not the driver are isolated. Briefly, tracer nucleic acid (cDNA or mRNA) from one cell population is allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences common to both cell populations. Hybrids between the tracer and driver, and all driver sequences, are removed in the subtraction step. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. The method described in this unit uses double‐stranded cDNA (ds cDNA) as both tracer and driver. Reciprocal subtractions are performed between two cell populations, A and B:
that is, genes preferentially expressed in A more than in B are isolated, as are genes expressed preferentially in B more than in A. The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction. The progress of subtraction is monitored by slot blot hybridization. Differentially expressed cDNA sequences are used to construct a subtracted cDNA library.
Differential Display of mRNA by PCR
Authors: Liang Peng , Pardee Arthur B.
Published: June 2004

Formerly UNIT , this unit describes how differential display techniques allow the identification and subsequent isolation of differentially expressed genes that requires no knowledge of sequences, but rather PCR amplification using arbitrary oligonucleotides and high resolution polyacrylamide gel electrophoresis.
Overview of RNA Interference and Related Processes
Authors: Baulcombe David
Published: June 2004

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.
RNA Interference in Caenorhabditis Elegans
Authors: Conte Darryl , Mello Craig C.
Published: June 2004

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.
Cloning of Small RNA Molecules
Authors: Pfeffer Sébastien , Lagos‐Quintana Mariana , Tuschl Thomas
Published: June 2004

Small RNAs that are derived from double‐stranded RNA 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 to directionally concatamerize the relatively short PCR products before cloning in order to increase the number of RNA sequences obtained per clone.
RNA Interference in Cultured Drosophila Cells
Authors: Worby Carolyn A. , Dixon Jack E.
Published: June 2004

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. To date, many Drosophila cell lines tested respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested have been efficacious at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss of function studies applicable to a wide variety of questions.
Identification of RNA Binding Proteins by UV Cross‐Linking
Authors: Luo Ming‐Juan , Reed Robin
Published: June 2004

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.
Purification of Functional RNA‐Protein Complexes using MS2‐MBP
Authors: Zhou Zhaolan , Reed Robin
Published: June 2004

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 microscopy.
Compendium of Drugs Commonly Used in Molecular Biology Research
Authors: Cole Nelson B.
Published: June 2004

This 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.
Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
Authors: Voytas Daniel , Ke Ning
Published: June 2004

This unit presents procedures for visualizing and quantitating radiolabeled proteins or DNA separated by polyacrylamide gel electrophoresis (PAGE) or affixed to filter membranes. Procedures for autoradiography and densitometry are provided, but even greater sensitivity can be achieved with fluorography or by the use of intensifying screens, both of which enhance the radioactive signal. Preflashing the film is described to increase the linear measurement range for weakly radioactive samples. Sensitivity and linear ranges of measurement can also be greatly extended by using a phosphor imaging system to quickly and easily quantify radioactive samples.
Toll Receptors
Authors: Takeda Kiyoshi , Akira Shizuo
Published: June 2004

This has led to the identification of several important roles for Toll receptors in mammals. This unit discusses mammalian Toll receptors (TLR1‐10) that have an essential role in the innate immune recognition of microorganisms. Also discussed are TLR‐mediated signaling pathways and antibodies that are available to detect specific TLRs.
Managing Immunocompromised Animals
Authors: Donovan John , Brown Patricia
Published: June 2004

This unit describes some commonly used methods for maintaining immunocompromised rodents, specifically mice, to prevent infections from adventitious pathogens. The basic protocol describes the microisolation housing unit, while alternate procedures cover individually ventilated isolator housing, laminar‐flow rack housing, flexible‐film isolator housing, and barrier facility housing.