bluuebunny/crossref_metadata_2025_split huggingface dataset:
One‐Dimensional Gel Electrophoresis of Proteins
Authors: Gallagher Sean R. , Smith John A. 
Published: June 2004

AbstractThis unit provides several methods for one‐dimensional gel electrophoresis of proteins. The basic protocol is the standard method used for gel electrophoresis under denaturing conditions. The first alternate protocol provides conditions used for nondenaturing gel electrophoresis. Gradient gels are described and resolve a much wider molecular‐weight range of proteins on a single gel. Minigels, both single‐concentration and gradient, are also described.
Isolation of Proteins for Microsequence Analysis
Authors: Moos Malcolm 
Published: June 2004

AbstractThe first basic protocol in this unit can be used to determine 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. Although the efficacy of this technique varies with the protein, it is possible to obtain useful sequence information starting with less than 50 pmol of the protein of interest. 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 in the second basic protocol. This method requires ˜200 pmol of protein for analysis.
Staining Proteins in Gels
Authors: Sasse Joachim , Gallagher Sean R. 
Published: June 2004

AbstractThis unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS‐polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.
Iodination of Soluble and Membrane‐Bound Proteins
Authors: Samelson L.E. 
Published: June 2004

AbstractCoupling of radioactive 125I to soluble proteins is a critical step in a number of immunoassays. Chloramine T‐catalyzed iodination of soluble proteins, such as immunoglobulins, is described in this unit. Protocols using lactoperoxidase‐catalyzed iodination of soluble or membrane‐bound proteins are also described.
Biosynthetic Labeling of Proteins
Authors: Bonifacino Juan S. 
Published: June 2004

AbstractBiosynthetic labeling techniques are commonly used in the study of biochemical properties, synthesis, processing, intracellular transport, secretion, and degradation of proteins. In this unit, protocols are described for biosynthetically labeling many secreted and membrane proteins. The describes short‐term labeling of cells in suspension (30 min to 3 hr) and an alternate protocol describes a modification of this procedure to be used for adherent cells. Alternate protocols for long‐term (3 to 24 hr) and pulse‐chase labeling of cells are also presented. Label incorporation can be determined as described in a support protocol. The cells used in these protocols can be either from suspension cultures or from single‐cell suspensions of spleen or thymus.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit 
Published: June 2004

AbstractThis unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. In the Basic Protocol, cells in culture are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady‐state level of incorporation. In the alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse‐chase labeling procedure is provided to analyze precursor‐product relationships. With the sequential pulse‐labeling method described here, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimum amount of labeled precursor by using the same batch of medium to pulse‐label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D. 
Published: June 2004

AbstractTreatment of cells with inhibitors of the enzymes that synthesize N‐linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N‐linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with [3H]mannose using TCA precipitation or endo H digestion. Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N‐linked oligosaccharide chains. A Support Protocol details a method for concentrating proteins by acetone precipitation.
Production of Anti‐Peptide Antisera
Authors: Carter John Mark 
Published: June 2004

AbstractThe protocols described in this unit can be used for generating and testing anti‐peptide antisera, assuming that a scientist has already selected and conjugated peptides for experimental vaccines. Described are immunization and bleeding of experimental animals, a simple ELISA for antiserum screening, and affinity purification of the serum, including preparation of columns.
Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries
Authors: Pinilla Clemencia , Appel Jon R. , Houghten Richard A. 
Published: June 2004

AbstractThis unit presents a combinatorial library method that consists of the synthesis and screening of mixture‐based synthetic combinatorial libraries (SCL) of peptide molecules. The protocols employ peptide SCL to identify peptides recognized by mAbs and T cells. The first protocol uses a positional scanning SCL (PS‐SCL) made up of hexapeptides to identify antigenic determinants recognized by mAbs. The 120 mixtures in the hexapeptide PS‐SCL are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide PS‐SCL to identify T cell peptide ligands. The 200 mixtures of the decapeptide PS‐SCL are tested for their ability to induce proliferation. Support protocols cover optimization of the assay conditions for each mAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide PS‐SCL, along with deconvolution approaches.
Agarose Gel Electrophoresis
Authors: Voytas Daniel 
Published: June 2004

AbstractAgarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5‐ to 25‐kb DNA fragments. The Basic Protocol in this unit can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light. Support protocols describe the use of midigels and minigels, as well as recommendations for photographing stained gels.
Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Authors: Moore David , Chory Joanne , Ribaudo Randall K. 
Published: June 2004

AbstractThis unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first basic protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper is also described and provides relatively high yields for fragments smaller than 2000 bp. Protocols are also provided for separating fragments larger than 1000 bp using low gelling/melting agarose gels and purified by phenol extraction, b‐agarase digestion of the gel, or via glass beads extraction. Sieving agarose gels can also be used to resolve very small DNA fragments. Removing linkers from a fragment using a column rather than a gel is included, followed by a method for estimating DNA concentrations in solution.
Southern Blotting
Authors: Brown Terry 
Published: June 2004

AbstractSouthern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer‐stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D. 
Published: June 2004

AbstractRestriction endonucleases recognize short DNA sequences and catalyze the cleavage of double‐stranded DNA at specific sites within or adjacent to the recognition sequences. This unit describes how to cleave DNA and refers the investigator to frequently updated manuals by endonuclease suppliers for the precise optimized conditions. Alternate procedures are given for digesting a DNA sample with several different enzymes and digesting multiple DNA samples with the same endonuclease.
Enzymatic Labeling of Nucleic Acids
Authors: Tabor Stanley , Boyle Ann 
Published: June 2004

AbstractExtensive knowledge of the enzymology involved in biosynthesis and degradation of nucleic acids has permitted the development of simple methods for labeling RNA and DNA with radioisotopes or biotin. These labeled probes are used primarily for hybridization to nucleic acid fragments of interest in a variety of applications. A complete description of the methods available for such labeling is beyond the scope of this manual, but contained within this unit are protocols for oligonucleotide‐primed synthesis of radiolabeled and biotinylated DNA probes, in vitro synthesis of radiolabeled RNA, and end labeling of synthetic oligonucleotide probes.
Analysis of RNA by Northern and Slot‐Blot Hybridization
Authors: Brown Terry 
Published: June 2004

AbstractSpecific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. The in this unit describes blotting and hybridization of RNA fractionated in an agarose/formaldehyde gel. This is arguably the quickest and most reliable method for northern analysis of specific sequences in RNA extracted from eukaryotic cells. An alternate protocol gives details of the glyoxal/DMSO method for denaturing gel electrophoresis, which may provide better resolution of some RNA molecules. A second describes slot‐blot hybridization of RNA samples, a rapid method for assessing the relative abundance of an RNA species in extracts from different tissues.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Okayama Hiroto 
Published: June 2004

AbstractThis unit contains two methods of calcium phosphate‐based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The uses a HEPES‐buffered solution to form a calcium phosphate precipitate that is directly layered onto the cells. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and is then dropped onto the cells. The alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, and may be helpful with linear or genomic DNA. Both methods of transfection require very high‐quality plasmid DNA, which can be prepared as described in the second . Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first .
Transfection Using DEAE‐Dextran
Authors: Selden Richard F 
Published: June 2004

AbstractTwo protocols for DEAE‐dextran transfection of cells are provided in this unit. The describes a procedure used to transfect adherent cells and the first presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second .
Use of Monoclonal Antibodies for Expression Cloning
Authors: Hollenbaugh Diane , Aruffo Alejandro , Jones Bryan , Linsley Peter 
Published: June 2004

AbstractThis unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell‐surface and intracellular proteins. The first protocol describes the cloning of cDNAs encoding cell‐surface antigens. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double‐stranded cDNA.
Quantitation of Rare DNAs by PCR
Authors: Coen Donald M. 
Published: June 2004

AbstractThis unit presents a protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it helps assess the presence of contaminating sequences, the bane of this kind of procedure. The DNA of interest is prepared and its concentration is determined. A known amount of this DNA is then mixed with two sets of oligonucleotide primers, one set specific for the DNA of interest (e.g., a virus) and the other set specific for an internal control (e.g., a single‐copy gene encoded by the host organism). The sequences between the primers are amplified, electrophoresed on a gel, transferred to a filter, and probed with oligonucleotides specific for each amplified product. The amounts of the amplified products from the DNA of interest can then be quantitated by comparison to the internal control. For simplicity, the protocol is written in terms of quantitating viral DNA molecules relative to host cellular sequences; however, it can be adapted readily for other applications.
Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
Authors: Max Edward E. , Mills Frederick C. , Chu Charles 
Published: June 2004

AbstractWhen a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)‐based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion‐circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.
Double‐Stranded DNA Sequence Analysis
Authors: Morrison Sherie L. 
Published: June 2004

AbstractThere are several different methods for analyzing the sequence of cloned DNA segments. For analysis of immunoglobulin variable (V) regions, double‐stranded DNA sequencing is routinely used. Although this method does not yield the longest DNA sequences, it is easy and provides sufficient information to determine the correct sequence. The protocols in this unit are for use with the Sequenase Version 2.0 kit from U.S. Biochemicals, which is based on dideoxy sequencing with a modified T7 DNA polymerase. The describes performing a sequencing reaction for cloned immunoglobulin V regions and running and analyzing a sequencing gel. A support protocol describes pouring and setting up the sequencing gel.
Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire
Authors: Currier Jeffrey R. , Robinson Mary Ann 
Published: June 2004

AbstractMeasuring the diversity of TCRs utilized by specific primary and memory T cell responses is critical to the fundamental understanding of regulation of the immune system. This unit describes the Spectratype/Immunoscope technique which permits an in depth analysis of the TCR repertoire present in a variety of biological samples from mice to humans. Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different CDR3 lengths from specific TCR variable region genes in a competitive manner. The PCR products are then resolved on polyacrylamide sequencing gels to reveal precise sizes in nucleotide base pairs. The unit also includes protocols that have been optimized to process and produce the starting materials required for spectratype analysis.
Preparation and Analysis of Phosphorylated Proteins
Authors: Siegel Jeffrey N. 
Published: June 2004

AbstractThe function of cells of the immune system is regulated in part by engagement of specific receptors on the cell surface by soluble ligands or ligands presented on the surface of other cells. One of the major mechanisms by which cell‐surface receptor engagement influences cellular function is by activating protein kinases and increasing the phosphorylation of critical cellular proteins. There are two major categories of protein kinases: serine/threonine kinases which phosphorylate both serine and threonine residues; and tyrosine kinases which phosphorylate tyrosine residues exclusively. By metabolically labeling cells with 32P, as described in this unit, the phosphorylation state of cellular proteins can be analyzed and changes occurring with receptor stimulation can be examined. Phosphoamino acid analysis is also described to determine whether serine, threonine, or tyrosine residues are phosphorylated, thereby indicating which category of protein kinase is responsible. A protocol for phosphopeptide mapping is provided to allow the investigator to produce a “fingerprint” of the peptides that are phosphorylated, providing information regarding the sites of phosphorylation.
Assessment of the Effects of Tyrosine Protein Kinase Inhibitors
Authors: Burkhardt Anne L. , Bolen Joseph B. 
Published: June 2004

AbstractThe tyrosine protein kinases are enzymes that are important in cellular signal transduction. Therefore, inhibition of TPKs provides an important means of investigating and potentially controlling many signaling pathways. The first basic protocol in this unit describes an assay of the inhibitory effects of TPK inhibitors in vitro on a specific TPK that has been immune‐precipitated from cell lysates. An assay of the effects of several TPK inhibitors on TPKs in vivo in activated cells is also provided. Although the example used here is a nonreceptor TPK, these protocols can be used to assay the effects of inhibitors on receptor TPKs as well.
Isolation and Quantitation of HIV in Peripheral Blood
Authors: Koup Richard A. , Ho David D. , Poli Guido , Fauci Anthony S. 
Published: June 2004

AbstractQuantitation of replication‐competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and therapy. In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring the tissue culture infectious dose (TCID) by an end‐point dilution method. A second basic protocol utilizes the PHA‐stimulated T cell blasts (activated T cells) in co‐culture with PBMC as described in the first basic protocol for the short‐term growth of HIV in vitro. An describes the accumulative method of determining 50% tissue culture infectious dose (TCID50) of HIV using the Reed‐Muench equation when multiple replicates of a given sample are employed in the assay. A consequence of HIV infection is the depletion of CD4+ target cells, evidenced by syncytia formation or single‐cell death; two support protocols detail the evaluation of these cytopathic effects.
Infection of CD4+ Primary Cell Lines and Induction of HIV
Authors: Poli Guido , Fauci Anthony S. 
Published: June 2004

AbstractChronically infected cell lines and clones are currently used as sources of virus to be used to infect other cell types as “factories” for large‐scale production of virus or viral components and as model systems for studies of the regulation of virus expression. In this unit, conditions for acute in vitro HIV infection are described. “Survivor” cells, resistant to the cytopathic effect of the virus, can be obtained from acute cultures. These survivor cells maintain an intact proliferative capacity and are considered to be chronically infected; their generation is described here. Finally, it is often desirable to study the effects of stimulants on HIV induction in these chronically infected cell lines; this procedure is detailed in a third protocol.