bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of Helper Virus in Retrovirus Stocks
Authors: Cepko Constance , Pear Warren 
Published: June 2004

AbstractHelper virus is a replication‐competent virus that is sometimes present in stocks of replication‐incompetent virus. There are several types of applications in which the presence of helper virus can be problematic. If animal infections are being done, helper virus can lead to leukemia, particularly if the infection is carried out pre‐ or neonatally. If retroviruses are being used for lineage analysis, helper virus may cause horizontal spread of the marker virus, creating false lineage relationships. This unit describes protocols for the detection of helper virus by a selectable marker assay, by rescue of an integrated provirus, or by measuring reverse transcriptase activity.
Two‐Dimensional Gel Electrophoresis Using the ISO‐DALT System
Authors: Adams Lonnie D. 
Published: June 2004

AbstractTwo high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) are combined in this unit to provide much greater resolution than either of the individual procedures. Solubilized proteins are first separated according to their isoelectric point by isoelectric focusing in a tube gel. This first dimension gel is then applied to the top of an SDS‐polyacrylamide slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are further separated on the basis of their molecular size. The ISO‐DALT system was specifically designed for running multiple high‐resolution two‐dimensional gels at one time.
Two‐Dimensional Gel Electrophoresis Using the O'Farrell System
Authors: Adams Lonnie D. , Gallagher (minigels) Sean R. 
Published: June 2004

AbstractTwo‐dimensional gel electrophoresis is the combination of two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first‐dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS‐slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in . For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two‐dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the support protocol.
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 staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining is 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.
Gel‐Filtration Chromatography
Authors: Hagel Lars 
Published: June 2004

AbstractGel filtration (GF) chromatography separates proteins solely on the basis of molecular size. Separation is achieved using a porous matrix to which the molecules, for steric reasons, have different degrees of access‐‐i.e., smaller molecules have greater access and larger molecules are excluded from the matrix. Hence, proteins are eluted from the GF column in decreasing order of size. This unit describes the experimental theory behind gel filtration and contains many useful tables listing the properties and characteristics of currently available matrices.
Ion‐Exchange Chromatography
Authors: Williams Alan , Frasca Verna 
Published: June 2004

AbstractIon‐exchange chromatography separates biomolecules on the basis of charge characteristics. Charged groups on the surface of a protein interact with oppositely charged groups immobilized on the ion‐exchange medium. This unit provides thorough explanations of the theory behind the technology, troubleshooting tips for making the procedure work best for you, and detailed listings of column matrices and separation properties.
Immunoaffinity Chromatography
Authors: Springer Timothy A. 
Published: June 2004

AbstractImmunoaffinity purification is a powerful technique for isolating proteins. Purifications of 10,000‐fold or more can often be achieved in one step. This unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. An alternate protocol describes the use of batch purification of antigens which can shorten the column loading time.
Metal‐Chelate Affinity Chromatography
Authors: Petty Kevin J. 
Published: June 2004

AbstractRecombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal‐chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. Techniques are discussed for creating a fusion protein consisting of the protein of interest with a histidine tail attached (for purification by MCAC). The basic protocol describes expression of histidine‐tail fusion proteins and their purification in native form by MCAC. Two alternate protocols describe purification of histidine‐tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid‐phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.
HPLC of Peptides and Proteins: Standard Operating Conditions
Authors: Boysen Reinhard I. , Hearn Milton T.W. 
Published: June 2004

AbstractThe standard operating conditions for the eight basic modes of HPLC are presented in this unit. 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. Procedures for multimodal column switching are also included, as are guidelines for a systematic approach to method development. Example separations help illustrate the procedures.The standard operating conditions for the eight basic modes of HPLC are presented in this unit.
Reversed‐Phase Isolation of Peptides
Authors: Henzel William J. , Stults John T. 
Published: June 2004

AbstractIn reversed‐phase HPLC, peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. Protocols describing the separation of peptides in 5‐ to 500‐pmol quantities via narrow‐bore (2‐mm‐i.d.) or microbore (1‐mm‐i.d.) columns, as well as for the separation of peptides in quantities <5 pmol are provided in this unit. Capillary HPLC columns require a gradient flow rate of 3 to 5 omponents present in a small sample prior to automated sequencing is possible via the procedures for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis
Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging
Authors: Zhang Ning , Chen Jin‐Long 
Published: June 2004

AbstractA protein molecule can be engineered to include a short stretch of residues corresponding to an epitope to facilitate its subsequent biochemical and immunological analysis; a technique often referred to as “epitope tagging.” This unit presents a protocol for small‐scale immunoprecipitation of epitope‐tagged recombinant proteins expressed in transiently transfected mammalian cells. The immunoprecipitant can then be analyzed by SDS‐PAGE. An immunoprecipitation protocol is also provided that has been optimized for use with a baculovirus overexpression system. An describes how multisubunit complexes can be assembled by starting with a core protein affixed to beads via an epitope tag, and adding the other members of the complex in a stepwise manner.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. , Springer Timothy A. 
Published: June 2004

AbstractImmunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro‐translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear‐cut explanation of the options for employing the technology.
Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes
Authors: Struhl Kevin 
Published: June 2004

AbstractThe availability of a cloned gene makes it possible to synthesize the encoded protein by in vitro transcription and translation. The basis of the method presented in this unit is to clone the protein‐coding sequences into a vector containing a promoter for SP6 or T7 RNA polymerase, to produce messenger RNA by transcribing the DNA template with this enzyme, and to synthesize the desired protein (often as an 35S‐labeled species) by translation of this mRNA in vitro. A major advantage of this method is that any desired mutant protein can be generated simply by altering the DNA template.
Capillary Electrophoresis of Proteins and Peptides
Authors: Burgi Dean , Smith Alan J. 
Published: June 2004

AbstractCapillary electrophoresis (CE) is a high‐resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides. As detailed here, CE can also be used to determine the isoelectric point of a protein, either in purified form or in a mixture, by focusing the sample in a pH gradient that is generated within the capillary during electrophoresis. In addition, CE can be used as a micropreparative technique, with either multiple separations that are pooled or a single, larger‐scale separation, for the isolation of peptides from a protease digestion (in much the same way that RP‐HPLC is currently used). In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.
Enzyme‐Linked Immunosorbent Assays (ELISA)
Authors: Hornbeck Peter , Winston Scott E. , Fuller Steven A. 
Published: June 2004

AbstractThis unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell‐surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid‐phase reactants. In the first four protocols, solid‐phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid‐phase reactants are cell‐associated molecules. In all protocols, the solid‐phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.
Immunization of Mice
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractIn the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen. Mice are immunized at 2‐ to 3‐week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.
Cloning of Hybridoma Cell Lines by Limiting Dilution
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractCloning by limiting dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90%
of the wells containing single clones are positive for antibody production.
Production of Monoclonal Antibody Supernatant and Ascites Fluid
Authors: Yokoyama Wayne M. 
Published: June 2004

AbstractProcedures detailing the production of monoclonal antibody supernatants, including the production of larger amounts (liters) are presented in this unit. An alternate protocol for large‐scale production of hybridomas or cells (e.g., for isolation of cellular proteins) involves a similar procedure. A method for producing and obtaining ascites fluid containing the monoclonal antibody is also presented.
In Vitro Antibody Production
Authors: Mond James J. , Brunswick Mark 
Published: June 2004

AbstractThis unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.
Identification of Polyol‐Responsive Monoclonal Antibodies for Use in Immunoaffinity Chromatography
Authors: Thompson Nancy E. , Burgess Richard R. 
Published: June 2004

AbstractOne of the limitations of immunoaffinity chromatography as been that high‐affinity antigen‐antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen‐antibody complexes can be dissociated in the presence of a combination of a low‐molecular‐weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein‐stabilizing. This type of antibody is designated “polyol‐responsive.“ These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the immunoaffinity purification of active, labile, multi‐subunit protein complexes.
DNase I Footprint Analysis of Protein‐DNA Binding
Authors: Brenowitz Michael , Senear Donald F. , Kingston Robert E. 
Published: June 2004

AbstractDeoxyribonuclease I (DNase I) protection mapping, or footprinting, is a valuable technique for locating the specific binding sites of proteins on DNA. The basis of this assay is that bound protein protects the phosphodiester backbone of DNA from DNase I‐catalyzed hydrolysis. Binding sites are visualized by autoradiography of the DNA fragments that result from hydrolysis, following separation by electrophoresis on denaturing DNA sequencing gels. Footprinting has been developed further as a quantitative technique to determine separate binding curves for each individual protein‐binding site on the DNA. For each binding site, the total energy of binding is determined directly from that site's binding curve. For sites that interact cooperatively, simultaneous numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative components of these energies.DNase I footprint titration is described in this unit and involves (1) preparation of a singly end‐labeled DNA restriction fragment, (2) equilibration of the protein with DNA, (3) exposure of the equilibrium mixture to DNase I, and (4) electrophoretic separation on gels of the denatured hydrolysis products, followed by autoradiography. A describes (1) densitometric analysis of the autoradiograms to obtain binding data and (2) numerical analysis of the binding data to yield binding curves and equilibrium constants for the interactions at each of the separate sites. An describes the qualitative use of footprinting to identify DNA‐binding proteins in crude extracts.
Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems
Authors: Chodosh Lewis A. 
Published: June 2004

AbstractShort fragments of DNA‐either natural or formed from oligonucleotides‐containing a high‐affinity site for a DNA‐binding protein provide a powerful tool for purification. The biotin/streptavidin purification system is based on the tight and essentially irreversible complex that biotin forms with streptavidin. In this procedure, a DNA fragment is prepared that contains a high‐affinity binding site for the protein of interest, and a molecule of biotinylated nucleotide is then incorporated into one of the ends of the DNA fragment. The protein of interest binds to the DNA, and then this complex binds (via the biotin moiety) to the tetrameric protein streptavidin. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently isolated by adsorption onto a biotin‐containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin‐containing resin. Proteins remaining in the supernatant are removed by washing, and the resin‐bound protein is then eluted with a high‐salt buffer. An alternate protocol describes a microcolumn method that is useful for larger volumes of biotin‐cellulose resin. This method is also used to elute the protein in as small a volume (i.e., as high a concentration) as possible. Another variation on the basic procedure is provided in which streptavidin‐agarose is employed.
Detection, Purification, and Characterization of cDNA Clones Encoding DNA‐Binding Proteins
Authors: Singh Harinder 
Published: June 2004

AbstractIn this unit, an appropriate recombinant clone is detected in an expression library with a DNA recognition‐site probe, purified, and shown to encode a DNA‐binding domain of defined sequence specificity. The strategy described below obviates purification of a sequence‐specific DNA‐binding protein for the purpose of isolating its gene; it simply requires a suitable cDNA library constructed in the expression vector lgt11 and a DNA recognition‐site probe. The basic protocol enables the detection and purification of clones encoding sequence‐specific DNA‐binding proteins. The alternate protocol describes a denaturation/renaturation procedure that can increase detection of certain clones. The support protocol provides a rapid means of characterizing the DNA‐binding activities of the proteins encoded by the cloned cDNAs.
Rapid Separation of Protein‐Bound DNA from Free DNA Using Nitrocellulose Filters
Authors: Papoulas Ophelia 
Published: June 2004

AbstractNitrocellulose binds proteins but not double‐stranded DNA. Use of radioactively labeled double‐stranded DNA fragments allows quantitation of DNA bound to the protein, permitting kinetic and equilibrium studies of DNA‐binding interactions. In the basic procedure, purified protein is mixed with double‐stranded DNA and then the mixture is filtered through nitrocellulose, allowing unbound DNA to pass through the filter while the protein (and any DNA interacting with it) is retained. When the binding site of a protein is unknown, the pure protein can be added to a mixture of fragments to select those fragments of DNA for which it has the greatest affinity. Specificity of binding can be influenced by the buffer conditions and filtering regimen. An is provided that creates conditions that disrupt weaker, presumably nonspecific binding interactions, while retaining the stronger binding interactions. The goal is to recover enough of a single input fragment to visualize by subsequent autoradiography. In some cases the quantitation (by scintillation counting) of DNA retained is not sufficient information. A describes how the DNA can be recovered from the filters for further analysis by gel electrophoresis or amplification and cloning.
Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography
Authors: Kerrigan Leslie A. , Kadonaga James T. 
Published: June 2004

AbstractAffinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence‐specific DNA‐binding properties. The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr‐activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.