bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunoblotting and Immunodetection
Authors: Gallagher Sean , Winston Scott E. , Fuller Steven A. , Hurrell John G.R. 
Published: June 2004

AbstractImmunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit presents procedures for electrophoretically transferring antigens from a denaturing polyacrylamide gel in a tank or a semidry transfer apparatus to a nitrocellulose, PVDF, or nylon membrane. The process can be monitored by reversible staining or by Ponceau S staining, both of which are described here. A protocol for blotting previously stained gels is also described. The transferred proteins are bound to the surface of the membrane, providing access for reaction with immunodetection reagents. All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent. After probing with the primary antibody, the membrane is washed and the antibody‐antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti‐IgG antibody (e.g., goat anti‐rabbit IgG). The enzymes are attached directly or via an avidin‐biotin bridge to the secondary antibody, and protocols are provided for both methods. Chromogenic or luminescent substrates are then used as described to visualize the activity. Finally, a method for stripping and reprobing membranes is presented.
Release of Saccharides from Glycoconjugates
Authors: Powell Leland D. , Varki Ajit P. , Freeze Hudson H. 
Published: June 2004

AbstractGlycosidases are specific enzymes that can partially or completely remove sugar chains from cell‐surface glycoconjugates. The enzymes can be exoglycosidases (which remove a terminal saccharide unit from an oligosaccharide chain), endoglycosidases (which cleave within an oligosaccharide chain, releasing an oligosaccharide fragment), or glycoamidases (which cleave between an oligosaccharide unit and its N‐linkage to a protein). Commonly used examples of each enzyme are presented in this unit. Sialidase digestion of purified proteins is described and an Alternate Protocol details the application of the technique to intact cell suspensions. A support protocol describes testing sialidase activity in a variety of buffers. Basic protocols are detailed for the digestion of intact glycoproteins and glycopeptides by the most common endoglycosidases and glycoamidases: Endoglycosidase H (Endo H), Endoglycosidase F2 (Endo F2), and Peptide:N‐glycosidase F (PNGase F). The applications described in the second and third support protocols employ these enzymes alone or as part of a sequential digestion. The second support protocol describes how to use partial digestions with one enzyme or sequential digestions with different enzymes to estimate the number or types of N‐linked carbohydrate chains on a protein. The third support protocols describes sample preparation and digestion followed by gel‐filtration chromatography to recover the released radiolabeled oligosaccharide chains for subsequent analysis.
Biotinylation and Analysis of Membrane‐Bound and Soluble Proteins
Authors: Lisanti M.P. , Sargiacomo M. 
Published: June 2004

AbstractCoupling of a biotin moiety to proteins allows sensitive detection of those proteins with a variety of commercially available avidin or streptavidin conjugates. This unit presents protocols for biotinylation of cell‐surface proteins and proteins in solution. A major advantage of these techniques is that they do not require use of radioisotopes (although use of radioiodinated streptavidin is optional for biotin detection).
Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
Authors: Grant Gregory A. 
Published: June 2004

AbstractAntibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The steps to produce an effective antibody include: (1) designing the peptide sequence based on the sequence of the protein; (2) synthesizing the peptide; (3) preparing the immunogen either by coupling the synthetic peptide to a carrier protein or through the use of a multiple antigenic peptide (MAP); (4) immunizing the host animal; (5) assaying antibody titer in the host animal's serum; and (6) obtaining the antiserum and/or isolating the antibody. This unit covers steps 1 and 3. Once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis. Also presented are methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides.
B Cell Epitope Mapping Using Synthetic Peptides
Authors: Carter John Mark , Loomis‐Price Larry 
Published: June 2004

AbstractSynthetic peptides are used for identification of functional B cell epitopes in antibody preparations. ELISA‐type assays are used to identify sequences of proteins comprising antibody‐binding regions. This unit describes three peptide display formats. Pepscan (pins) and SPOTs (cellulose membranes) may be used as solid‐phase support media for peptide synthesis, followed by ELISA directly on the resulting peptide array. Alternatively, peptides may be cleaved from the array and tested in a standard microplate‐based antibody capture ELISA format. The discussion includes choosing the peptide sequences by length and overlap, as well as determination of the minimum essential sequence for antibody binding. This method is highly effective for continuous epitopes and is often also useful for discontinuous epitopes.
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David 
Published: June 2004

AbstractThis unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. The , using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations <1 mg/ml. Isopropanol may also be used to precipitate DNA, as described in an alternate protocol. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads, and is described here. These protocol may also be used for purifying RNA. The final protocols provide modifications to the that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing ow‐molecular‐weight oligonucleotides and triphosphates.
Hybridization Analysis of DNA Blots
Authors: Brown Terry 
Published: June 2004

AbstractThe principle of hybridization analysis is that a single‐stranded DNA or RNA molecule of defined sequence (the probe) can base‐pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs. Experimentally, the analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. Hybridization analysis is sensitive and permits detection of single‐copy genes in complex genomes. This unit presents a basic procedure for hybridization analysis with a radiolabeled DNA probe. Despite its lack of embellishment, the protocol gives acceptable results with Southern blots on nitrocellulose and nylon (uncharged and charged) membranes. An describes a similar method for probing DNA blots with a radiolabeled RNA probe. A for stripping blots to ready them for reprobing is also provided.
Preparation of RNA from Tissues and Cells
Authors: Ribaudo Randall , Gilman Michael , Kingston Robert E. , Choczynski Piotr , Sacchi Nicoletta 
Published: June 2004

AbstractMost procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.
Transfection by Electroporation
Authors: Potter Huntington 
Published: June 2004

AbstractElectroporation yields a high frequency of permanent transfectants, has a high efficiency of transient gene expression, and can be easier to carry out than alternative techniques. Electroporation makes use of the fact that the cell membrane acts as an electrical capacitor which is unable (except through ion channels) to pass current. Subjecting membranes to a high‐voltage electric field results in their temporary breakdown and the formation of pores that are large enough to allow macromolecules (as well as smaller molecules such as ATP) to enter or leave the cell. The reclosing of the membrane pores is a natural decay process which is delayed at decreased temperatures. This unit presents the procedures which can be used for both transient and stable transfections.
Stable Transfer of Genes into Mammalian Cells
Authors: Kingston Robert E. 
Published: June 2004

AbstractAnalysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA, so a dominant selectable marker is used to permit isolation of stable transfectants. Appropriate selection conditions for the parental cell line are first determined, and the gene to be studied is then transfected into that cell line with a gene that expresses a selectable marker. The cells are allowed to grow under selection for approximately 10 doublings before individual colonies are picked and expanded into cell lines. This unit includes conditions for six markers commonly used for selection of mammalian cells. An alternative, specialized procedure for transferring immunoglobulin chains into lymphoid cells is presented in Unit .
Transfection of Lymphoid Cells
Authors: Morrison Sherie L. 
Published: June 2004

AbstractUsing available vectors, a single immunoglobulin chain or both heavy (H) and light (L) chains can be simultaneously transferred to recipient myeloma cells. Several methods can be used to prepare stable transfected lymphoid cells. The in this unit describes protoplast fusion, which is suitable for certain lymphoid cells but tends to be more cumbersome than electroporation or liposome‐mediated transfection, which are described in the two alternate protocols. All three techniques may be adapted for stable transfection of other eukaryotic cell lines.
Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems
Authors: Swift Susan , Lorens James , Achacoso Philip , Nolan Garry P. 
Published: June 2004

AbstractThis unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate‐mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion‐containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be “pseudotyped” with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV‐G‐pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.
Construction of Immunoglobulin Fusion Proteins
Authors: Hollenbaugh Diane , Aruffo Alejandro 
Published: June 2004

AbstractRecombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.
Phage Display of Single‐Chain Antibody Constructs
Authors: Benhar Itai , Reiter Yoram 
Published: June 2004

AbstractPhage display is based on expressing recombinant proteins fused to a phage coat protein. The genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. With phage display, tailor‐made proteins (fused peptides, antibodies, enzymes, DNA‐binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity and specificity of binding. This unit provides protocols for the construction of, and selection from antibody phage display libraries, together with useful approaches for the analysis of phage antibodies. This unit focuses on the construction of immune libraries where the sources for antibody genes are spleens of immunized mice and the isolation and initial characterization of single‐chain antibodies (scFvs) from such libraries.
cDNA Amplification Using One‐Sided (Anchored) PCR
Authors: Dorit Robert L. , Ohara Osamu 
Published: June 2004

AbstractThis unit presents a modification of PCR, called anchored PCR, that allows amplification of full‐length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.
Molecular Analysis of Mouse T Cell Receptor Expression Using PCR
Authors: Fox Casey J. , Danska Jayne S. 
Published: June 2004

AbstractThis unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra‐lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR‐amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.
Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression
Authors: Young Howard A. , Subleski Jeffrey J. , Krebs Stephanie M. 
Published: June 2004

AbstractThe multiprobe RNase protection assay enables investigators to monitor RNA expression of 8‐12 genes with as little as 1 µg of total RNA. The commercial availability of numerous multi‐gene template sets makes this assay practical for all basic research programs.
Immune‐Complex Assays for Tyrosine Protein Kinases
Authors: Burkhardt Anne L. , Bolen Joseph B. 
Published: June 2004

AbstractTyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune‐complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS‐PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high‐throughput assay that evaluates only exogenous substrate phosphorylation.
Analyzing Mitogen‐Activated Protein Kinase (MAPK) Activities in T Cells
Authors: Whitmarsh Alan J. , Davis Roger J. 
Published: June 2004

AbstractThe recognition of peptide antigens by T cells through their antigen receptors (T cell receptors, or TCR), together with the ligation of additional surface molecules called costimulatory receptors, rapidly induces interactive signaling pathways that lead to transcriptional initiation at genes such as that of the autocrine growth factor interleukin 2 (IL‐2). Activation of the ERK and JNK subfamilies of MAPK mediates some of these signals. This unit presents procedures for a solid‐phase kinase assay and immune‐complex kinase assay to measure JNK and ERK activities, respectively, in T cells that have been appropriately stimulated. Also described is a procedure for preparing GSThyphen;cJun/GSH‐Sepharose beads needed in the solid‐phase JNK protein kinase activity assay.
Key Molecules Involved in Receptor‐Mediated Lymphocyte Activation
Authors: O'Shea John J. , Johnston James A. , Kehrl John , Koretzky Gary , Samelson Lawrence E. 
Published: June 2004

AbstractThis unit, along with Unit 11.9B, provides a summary of our current knowledge about various signaling pathways critical to the function of immune cells. Here, our understanding of T cell receptor (TCR)‐ and B cell receptor (BCR)‐mediated signaling is summarized. A schematic representation of immunologically relevant cytokine receptors and the Janus Family Kinases (JAKs) that is activated through these receptors is provided, along with details about molecules involved in interleukin 2 mediated signal transduction.
Signal Transduction During Natural Killer Cell Activation
Authors: Chini Claudia C.S. , Leibson Paul J. 
Published: June 2004

AbstractNatural killer (NK) cells are a subpopulation of lymphocytes that can mediate cytotoxicity of certain tumor cells, virus‐infected cells, and normal cells. In addition to their cytotoxic potential, NK cells secrete a variety of cytokines and chemokines that can modulate the function, growth, and differentiation of other immune cells. These different responses are initiated by the interaction of specific NK surface receptors with defined soluble or cell‐associated ligands. There are several different types of receptors on the NK cell surface including “triggering” receptors, adhesion molecules, cytokine receptors, and MHC‐recognizing killer‐cell inhibitory receptors. The functional response of an NK cell is the result of the integration of signals transduced by these different types of receptors. Some of these signaling pathways are similar to other lymphoid cells, but there are also unique features employed by NK cells. This overview focuses on receptor‐initiated signaling pathways that modulate NK functions.
Apoptosis Signaling Pathways
Authors: Siegel Richard M. , Lenardo Michael J. 
Published: June 2004

AbstractIn the past few years much has been learned about the molecular signals governing apoptosis, or programmed cell death, in lymphocytes and other cells of the immune system. Two major pathways, active and passive apoptosis, have been identified and are discussed in this overview. Active apoptosis, also termed propriocidal cell death or antigen‐induced cell death, occurs when cells are stimulated through a family of TNF‐related receptors termed death receptors. These receptors are up‐regulated in activated lymphocytes and trigger apoptosis chiefly in effector cells that have been recently stimulated through the antigen receptor. Passive or lymphokine withdrawal apoptosis occurs when activated lymphocytes are deprived of essential growth cytokines and does not require death receptors. Cell death initiated by either pathway is carried out by a unique family of intracellular cysteine proteases, the caspases. Defects in each of these pathways produce distinct pathologies.
General Guidelines for Experimenting with HIV
Authors: Byers Karen B. , Engelman Alan , Fontes Benjamin 
Published: June 2004

AbstractPerforming experiments using human immunodeficiency virus (HIV)‐infected materials represents a potential biological hazard for the investigator. An intensive training program for laboratory personnel must always precede the actual execution of research involving manipulation of HIV. In addition, appropriate sterile tissue culture techniques are absolute prerequisites for producing meaningful experiments and for achieving safe working conditions. In most circumstances more than one investigator uses the same HIV research facility; therefore, careful training in biosafety is mandatory not only for self‐protection, but for the safety of other investigators as well. The first protocol in this unit establishes a general framework for working safely with HIV and the second describes the proper storage of HIV stocks and test supernatants.
Detection Assays for HIV Proteins
Authors: Ostrowski Mario A. , Chun Tae‐Wook , Stanley Sharilyn K. , Fauci Anthony S. 
Published: June 2004

AbstractThis updated unit describes four assays for detection of HIV proteins including: (1) a quantitative ELISA, (2) a quantitative immunoblotting assay, (3) a qualitative immunofluorescence assay, and (4) a functional assay to measure virus‐associated reverse transcriptase activity as an indicator of viral production.