bluuebunny/crossref_metadata_2025 huggingface dataset:
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R.
Published: June 2004

Electrophoresis is used to separate complex mixtures of proteins, (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several alternate protocols are provided for specific applications. The first two alternate protocols cover electrophoresis of peptides and small proteins, separations that require modification of standard buffers: either a Tris‐tricine buffer system, or modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and electrophoresis of various types of gels: ultrathin gels, multiple single‐concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.
Overview of Digital Electrophoresis Analysis
Authors: Medberry Scott , Gallagher Sean , Moomaw Butch
Published: June 2004

Gel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide‐based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single‐gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image‐capture device, and the images can be easily manipulated using image analysis software. This overview presents reasons for digital documentation and analysis, defines some important key terms for imaging, explains the capture process and reviews the devices used for image capture, and provides an introduction to the software and methods used for one‐ and two‐dimensional digital image analysis.
Detection of Proteins on Blot Transfer Membranes
Authors: Sasse Joachim , Gallagher Sean R.
Published: June 2004

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.
HPLC of Peptides and Proteins: Preparation and System Set‐Up
Authors: Boysen Reinhard I. , Hearn Milton T.W.
Published: June 2004

The eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size‐exclusion chromatography (HP‐SEC), ion‐exchange chromatography (HP‐IEX), normal phase chromatography (HP‐NPC), hydrophobic interaction chromatography (HP‐HIC), reversed‐phase chromatography (RP‐HPLC), hydrophilic interaction chromatography (HP‐HILIC), immobilized metal ion affinity chromatography (HP‐IMAC), and biospecific/biomimetic affinity chromatography (HP‐BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP‐MMC), charge transfer chromatography (HP‐CTC), or ligand‐exchange chromatography (HP‐LEC) are described. The unit includes overviews of the system requirements and planning strategies for set‐up of the systems.
Metabolic Labeling with Amino Acids
Authors: Bonifacino Juan S.
Published: June 2004

Metabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This update focuses on pulse‐labeling and pulse‐chase experiments done with
[35S]‐methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S‐labeled compounds.
Isolation of Proteins for Microsequence Analysis
Authors: Moss Malcolm
Published: June 2004

This unit presents a method for determining the amino‐terminal sequence of polypeptides or proteins. It is particularly appropriate for large fragments of insoluble or hydrophobic proteins or proteins that cannot be purified to >90% molar purity without electrophoresis. If the protein is blocked at the amino terminus, chemical cleavage or partial enzymatic digestion must be performed prior to electrophoresis. Upon isolation, the internal amino acid sequence is analyzed as described.
Overview of Peptide and Protein Analysis by Mass Spectrometry
Authors: Carr Steven A. , Annan Roland S.
Published: June 2004

The goals of this unit are to familiarize peptide and protein chemists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. The emphasis here is on established tools and techniques that can realistically be used for problem solving. As a result, many useful instruments and techniques employed by the trained mass spectroscopist are not discussed here, because they do not satisfy these criteria or are not yet widely available. A few of these are briefly mentioned at the end of this introductory overview. The overview concludes with a tutorial discussion on the meaning and practical importance of a number of fundamental experimental and performance‐related issues that have important implications for interpretation and use of mass spectral data, such as mass accuracy and resolution.
Conjugation of Enzymes to Antibodies
Authors: Winston Scott E. , Fuller Steven A. , Evelegh Michael J. , Hurrell John G.R.
Published: June 2004

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen‐specific monoclonal or polyclonal antibody in which neither the antigen‐combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross‐linking HRPO, urease or alkaline phosphatase to immunoaffinity‐purified monoclonal or polyclonal antibodies (IgG).
Isotype Determination of Antibodies
Authors: Hornbeck Peter , Fleisher Thomas A. , Papadopoulos Nicholas M.
Published: June 2004

Frequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti‐immunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.
Preparation of Myeloma Cells
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R.
Published: June 2004

In this unit, myeloma cells are cultured to ensure their sensitivity to the HAT selection medium used after cell fusion. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion. A support protocol is provided to determine the number of viable cells present in the cell culture.
Preparation of Mouse Feeder Cells for Fusion and Cloning
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R.
Published: June 2004

To maximize the yield of hybrids from fusion and cloning procedures, feeder cells are required to be cocultured with the hybrids, while hybrid cell density is low. Mouse peritoneal cells, most of which are macrophages, have been found to be convenient and effective feeder cells which are a source of soluble growth factors for hybridoma cells.
Fusion of Myeloma Cells with Immune Spleen Cells
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R.
Published: June 2004

Liquid nitrogen storage is the method of choice for long‐term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.
Production of Polyclonal Antisera
Authors: Cooper Helen M. , Paterson Yvonne
Published: June 2004

In this unit, protocols describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. A support protocol presents a method for preparing serum from blood.
Determination of the Specific Antibody Titer
Authors: Cooper Helen M. , Paterson Yvonne
Published: June 2004

The amount of specific antibody present in polyclonal antiserum, ascites fluid, or hybridoma supernatant can be quantitated by either solid‐phase radioimmunoassay (RIA) or by direct ELISA. In the solid‐phase assay described here, serially diluted antiserum is incubated in microtiter wells previously coated with the relevant antigen. Bound antibody is detected by employing 125I‐labeled anti‐immunoglobulin antibodies. The amount of specific antibody in the antiserum is then determined from a standard curve generated with a specific antibody of known concentration. The unknown antiserum and the standard antibody are assayed in parallel. The support protocols describe the chloramine T and IODO‐GEN procedures for radioiodination of the anti‐immunoglobulin reagent. The use of the solid‐phase RIA procedure to determine the light‐chain and heavy‐chain isotypes present in polyclonal antisera and fluids containing monoclonal antibodies is also described.
Genome‐Wide Transposon Mutagenesis in Yeast
Authors: Kumar Anuj , Snyder Michael
Published: June 2004

This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon‐mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon‐tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon‐mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).
Preparation of Yeast DNA
Authors: Hoffman Charles S.
Published: June 2004

Molecular studies in yeast often require the isolation of both plasmid and chromosomal yeast DNA. Plasmid DNA is used in the transformation of E. coli, whereas chromosomal DNA is used for Southern hybridization analysis, in vitro amplification by the polymerase chain reaction (PCR), or cloning of integrated plasmids. This unit presents two variations of the “smash and grab” protocol that produce suitable DNA for all these applications. These protocols work for both Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Preparation of Yeast RNA
Authors: Collart Martine A. , Oliviero Salvatore
Published: June 2004

This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample‐to‐sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.
S. pombe Strain Maintenance and Media
Authors: Forsburg Susan L.
Published: June 2004

Fission yeast can be grown and maintained using similar culture methods to those employed for budding yeast. The optimal media for S. pombe is somewhat different than that employed for S. cerevisiae, although budding yeast media can be used if necessary. Good sterile technique is essential to prevent contamination by airborne molds or bacteria.
In situ Hybridization and Detection Using Nonisotopic Probes
Authors: Knoll Joan H.M. , Lichter Peter
Published: June 2004

Nonisotopic in situ hybridization can be used to determine the cellular location and the relative levels of expression of specific transcripts within cells and tissues. RNA in prepared specimens is hybridized with a probe labeled nonisotopically with biotin or digoxigenin, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in addition to techniques for amplification of weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here.
Principles and Application of Fluorescence Microscopy
Authors: Coling Donald , Kachar Bechara
Published: June 2004

This overview discusses the principle of fluorescence along with practical discussions of fluorescent molecular probes, filters and filter sets, multiband filters and multi‐dye fluorescence, light sources, objective lenses, image resolution and the point‐spread function, fluorescence microscopy of living cells, and immunolabeling.
Measurement of In Situ Hybridization
Authors: Hoover Shelley B. , Fox Cecil H. , Bahre Jim , Cottler‐Fox Michele , Sung Cynthia
Published: June 2004

Hybridization of labeled specific molecular probes to nucleic acids in tissues allows geometric and functional location of gene expression or of foreign genome sequences. Estimates of amounts and location of target nucleic acid sequence can be made with phosphor storage imaging and molecular controls.
Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
Authors: Martin David , Lenardo Michael
Published: June 2004

As programmed cell death (PCD), or apoptosis, has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. Necrosis refers to the morphology usually associated with accidental cell death, while apoptosis is seen when cell death is programmed or physiologically regulated. This unit presents a set of assays for these purposes, many of which are technically very simple. Featured in this unit is the TUNEL method of detecting cells that exhibit DNA fragmentation, which can also be performed on tissue sections to locate apoptotic cells in situ.
Enzymatic and Chemical Cleavage of Fusion Proteins
Authors: LaVallie Edward R. , McCoy John M. , Smith Donald B. , Riggs Paul
Published: June 2004

This unit provides protocols for some commonly used methods of site‐specific cleavage of fusion proteins. The first three protocols describe enzymatic cleavage of proteins using proteases (factor Xa, thrombin, and enterokinase) that display highly restricted specificities, which greatly decrease the likelihood that unwanted secondary cuts will occur. Three additional protocols describe specific cleavage of fusion proteins with chemical reagents (cyanogen bromide, hydroxylamine, and low pH) as an alternative to enzymatic cleavage.
Expression and Purification of Maltose‐Binding Protein Fusions
Authors: Riggs Paul
Published: June 2004

This unit describes the procedure for subcloning the sequence encoding the protein of interest into an maltose‐binding protein (MBP) vector, and expressing and purifying the fusion protein from the cytoplasm. MBP vectors include a sequence that encodes the four‐amino‐acid recognition site for the specific protease factor Xa. The site is placed so it can be used to separate the protein of interest from MBP after affinity purification. A support protocol provides a pilot experiment for analyzing the solubility, affinity for the amylose resin, and export of a particular fusion protein. An alternate protocol gives instructions for purifying a fusion protein from the periplasm for fusions that are made in the signal sequence vector and are exported. Additional support protocols detail two different chromatographic methods for separating the protein of interest from MBP after factor Xa cleavage.
Overview of the Baculovirus Expression System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying
Published: June 2004

Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post‐translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.
Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying
Published: June 2004

This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild‐type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.
Expression and Purification of Recombinant Proteins Using the Baculovirus System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying
Published: June 2004

This unit describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production , how to maximize and scale up recombinant protein production, and how to purify recombinant proteins.
Transient Expression of Proteins Using COS Cells
Authors: Aruffo Alejandro
Published: June 2004

This unit describes the use of COS cells to efficiently produce a desired protein in a short period of time. These cells express high levels of the SV40 large tumor (T) antigen, which is necessary to initiate viral DNA replication at the SV40 origin. Each COS cell transfected with DNA encoding a cell‐surface antigen (in the appropriate vector) or cytoplasmic protein will express several thousand to several hundred thousand copies of the protein 72 hr posttransfection. If the transfected DNA encodes a secreted protein, up to 10 mg of protein can be recovered from the supernatant of the transfected COS cells 1 week posttransfection. COS cell transient expression systems have also been used to screen cDNA libraries, to isolate cDNAs encoding cell‐surface proteins, secreted proteins, and DNA binding proteins, and to test protein expression vectors rapidly prior to the preparation of stable cell lines.
Overview of the Vaccinia Virus Expression System
Authors: Moss Bernard , Earl Patricia L.
Published: June 2004

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+
kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and “appropriate” transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.
Generation of HIV‐1‐Based Lentiviral Vector Particles
Authors: Ramezani Ali , Hawley Robert G.
Published: June 2004

This unit presents protocols outlining the methodology and techniques involved in the construction and application of HIV‐1‐based lentiviral vector systems. Also described are procedures that can be used to concentrate and purify high‐titer recombinant lentiviral vector preparations, as well as protocols for transduction of adherent and suspension cells.
Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification
Authors: Esko Jeffrey D.
Published: June 2004

In this unit, protocols describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. A support protocol presents a method for preparing serum from blood.
Direct Chemical Analysis of Glycoconjugates for Carbohydrates
Authors: Manzi Adriana , Esko Jeffrey
Published: June 2004

This unit presents protocols for detection of different sugars bound to glycoconjugates. The solution containing the carbohydrate material is treated with a specific reagent, generating a colored reaction product that can be detected spectrophotometrically. For each assay, the absorbance of the colored solution is proportional to the amount of sugar present in the glycoconjugate.
Detection of Individual Glycosylation Sites on Glycoproteins
Authors: Powell Leland D.
Published: June 2004

In this unit, glycopeptides generated by endopeptidase digestion are first separated by reversed‐phase chromatography. The presence of hydrophilic, negatively charged oligosaccharides shortens retention times, causing glycopeptides to elute in considerably broader peaks than do peptides, so by following the elution profile either radiochemically or colorimetrically, the peaks corresponding to unique glycopeptides can be identified. With proper controls, the number of peaks will correspond to the number of different glycosylation sites. The eluted fractions are suitable for analysis by lectin chromatography, and the peptide sugar linkage can be defined either by endoglycosidase digestion or chemical cleavage. Oligosaccharides freed from the peptide according to the methods described in this unit can be characterized by size or charge, techniques not generally applicable with glycopeptides.
β‐Elimination for Release of O‐GalNAc‐Linked Oligosaccharides from Glycoproteins and Glycopeptides
Authors: Fukuda Minoru
Published: June 2004

This unit describes release of oligosaccharides that are attached to polypeptides through an N‐acetylgalactosamine (GalNAc) linkage to the hydroxyl groups of serine or threonine. The b‐elimination procedures described here can be used to recover the oligosaccharide chains (also called glycans) and/or identify the serine or threonine residues involved in the linkage. A b‐elimination method employing sodium borohydride (NaBH4) and alkaline conditions is described, and an alternative method is also presented in which only alkaline conditions are used without a reducing agent. Another alternative protocol uses sodium sulfite.
Acid Hydrolysis for Release of Monosaccharides
Authors: Manzi Adriana
Published: June 2004

The first step in obtaining a compositional analysis of a glycoconjugate is the release of the individual monosaccharide constituents. Mild acid hydrolysis to release fucosyl residues from glycoconjugates is described in this unit, in addition to mild acid hydrolysis to release sialic acids from glycoconjugates, along with dialysis and column chromatography procedures to purify the sialic acids. Strong acid hydrolysis to release all monosaccharides from glycoconjugates is also detailed.
Enzymatic Release of Oligosaccharides from Glycolipids
Authors: Manzi Adriana E.
Published: June 2004

Selective cleavage of the oligosaccharide moiety of glycolipids for further structural analysis can be achieved by means of endoglycoceramidase (EGCase), an enzyme specific for the linkages between oligosaccharide and ceramide residues in glycolipids. The method described here can be used for analysis of glycolipids present in macroscopic amounts or for glycolipids that have been radiolabeled in their carbohydrate moiety.
Endo‐β‐Galactosidases and Keratanase
Authors: Fukuda Michiko N.
Published: June 2004

This overview covers the endo‐β‐galactosidases; enzyme is capable of hydrolyzing a wide range of glycoconjugates. Endo‐β‐galactosidases from numerous sources are discussed in terms of their substrate specificities and substrates, as well as their practical research applications.
Fractionation and Analysis of Neutral Oligosaccharides by HPLC
Authors: Hayes Bradley K. , Varki Ajit
Published: June 2004

This unit describes the fractionation and analysis of neutral oligosaccharides by high‐performance liquid chromatography (HPLC) on bonded amine columns. Separation is based upon hydrogen bonding between the NH2 groups of the column and the hydroxyl groups of the oligosaccharides. A support protocol describes the reduction and desalting of neutral oligosaccharides with sodium borohydride.
Nitrous Acid Degradation of Glycosaminoglycans
Authors: Conrad H. Edward
Published: June 2004

Glycosaminoglycans (GAGs) are made up of disaccharide units that are distinguished from each other by the monosaccharide units of which they are composed and by the degree and position of sulfation. These disaccharide units represent the monomeric units of the GAG; thus, measurement of the disaccharide composition of a GAG represents the first step in the characterization of the polymer. In this unit, cleavage of the glycosidic bonds of the N‐sulfated GlcN residues in heparin and heparan sulfate is described, in addition to cleavage of the bonds between the N‐acetylated amino sugar residues in heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate, and hyaluronic acid. Using these procedures involving, all GAGs can be converted completely to their constituent disaccharides and reduced with NaB[3H]4) to yield labeled disaccharides that can be assayed qualitatively or quantitatively. The procedure may also be used to analyze metabolically labeled GAGs (with or without the use of NaB[3H]4).
Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans
Authors: Conrad H. Edward
Published: June 2004

Glycosaminoglycans (GAGs) are converted to disaccharides by various methods. This unit describes separation of individual disaccharides by paper chromatography or paper electrophoresis or high‐performance liquid chromatography (HPLC). Lyase‐released disaccharides can also be monitored by UV absorbance. Support protocols describe mild conditions for reduction of alkali‐labile disaccharides obtained by cleavage of GAGs with lyases, scintillation counting of samples obtained from HPLC separation of radiolabeled saccharides, and calculations for the quantitation of disaccharides.
Analysis of Sulfate Esters by Solvolysis or Hydrolysis
Authors: Freeze Hudson H.
Published: June 2004

Sulfate esters are found on N‐ and O‐linked sugar chains or glycosaminoglycan (GAG) chains. Few sulfatases are available that can enzymatically remove them, so chemical procedures must be used. These procedures rely on the differential sensitivity of sulfates located in different linkages on the sugar. In comparison to the conditions used for enzymatic digestion, those used for chemical digestion are very harsh and cannot be used on protein‐bound carbohydrates (except for analytical purposes, as described here). With protein‐bound carbohydrates, for preparative purposes, the chains must first be released. This unit describes release of sulfate esters by solvolysis along with a method for monitoring the efficiency of the solvolysis reaction. An alternate procedure provides a scale‐up method for using the solvolysis reaction with large amounts of material. Also presented are techniques for both acid and basic hydrolysis to release sulfate esters.
Permeabilization Strategies to Study Protein Phosphorylation
Authors: Carter A. Nigel
Published: June 2004

This 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

This 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

Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible 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

Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild‐type kinases. These engineered kinases, which are referred to as “analog‐sensitive” or “–as” alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by –as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild‐type kinase genes with engineered alleles, the in vivo activity of a –as kinase can be reversibly blocked with an allele‐specific inhibitor. Thus, analog‐sensitive kinases can be effective tools for 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

Phage‐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

Coprecipitation of proteins from whole‐cell extracts is a valuable approach to test for physical interactions between proteins of interest. This unit describes basic approaches to immunoprecipitating tagged proteins from whole‐cell extracts. The approaches described can be adapted for other systems. In a typical experiment, as described here, cells are lysed and a whole‐cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid‐phase affinity matrix and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope‐tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high 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

Far 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

This unit describes methodology for using micrococcal nuclease to investigate the presence of nucleosomes at a particular location in chromatin and to map the positions of nucleosomes at various levels of resolution. The approaches are readily adaptable to other probes of chromatin structure that cause DNA cleavage. Results obtained from such chromatin studies provide a structural view of the molecular environment of gene in their native context in cells.
DNase I and Hydroxyl Radical Characterization of Chromatin Complexes
Authors: Vitolo Joseph M. , Thiriet Christophe , Hayes Jeffrey J.
Published: June 2004

The 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

Because 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

Nucleic 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

The 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

Building 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

Homozygous 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

The 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.
Overview of Agents from Combinatorial Nucleic Acid and Protein Libraries
Authors: Elbing Karen , Brent Roger
Published: June 2004

This unit will be published in an upcoming supplement.
In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
Authors: Jhaveri Sulay D. , Ellington Andrew D.
Published: June 2004

In 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

Peptide 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

mRNA 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

Laser 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

A 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.