bluuebunny/crossref_metadata_2025_split huggingface dataset:
Denaturing Gel Electrophoresis for Sequencing
Authors: Slatko Barton E. , Albright Lisa M. 
Published: June 2004

AbstractThe accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40‐cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge‐shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the ‐‐inclusion of formamide in the sequencing gel‐‐is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.
Linker‐Scanning Mutagenesis of DNA
Authors: Greene John M. 
Published: June 2004

AbstractTwo protocols are described in which clusters of point mutations are introduced throughout a sequence of interest that has been cloned into a plasmid vector. The first protocol uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides are synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence actually provides the desired clusters of point mutations as it is moved or “scanned” across the region by its position at the varied endpoints of the deletion mutation series. An makes use of site‐directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the target region.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Rose John K. 
Published: June 2004

AbstractThis unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES‐buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.
Nonisotopic Assays for Reporter Gene Activity
Authors: Brasier Allan R. , Fortin John J. 
Published: June 2004

AbstractThis unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the β‐galactosidase expression plasmid. Both of these chemiluminescent assays have the advantages of high sensitivity and broad linear range. In the chemiluminescent detection procedures given in this unit, both luciferase activity and β‐galactosidase activity can be measured with either a luminometer or a scintillation counter.
One‐Dimensional Electrophoresis Using Nondenaturing Conditions
Authors: Gallagher Sean R. 
Published: June 2004

AbstractNondenaturing or “native” electrophoresis (i.e., electrophoresis in the absence of denaturants such as detergents and urea) is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Because mobility depends on the size, shape, and intrinsic charge of the protein, nondenaturing electrophoresis provides a set of separation parameters distinctly different from mainly size‐dependent denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and charge‐dependent isoelectric focusing. Two protocols are presented in this unit. Continuous PAGE is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH. The discontinuous procedure is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.
Immunoblotting and Immunodetection
Authors: Gallagher Sean , Winston Scott E. , Fuller Steven A. , Hurrell John G.R. 
Published: June 2004

AbstractImmunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody‐antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti‐IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.
Protein Identification and Characterization by Mass Spectrometry
Authors: Patterson Scott D. 
Published: June 2004

AbstractThis overview describes some of the new technologies that can be employed to facilitate rapid identification and characterization of proteins, including the use of correlative approaches for protein identification, rapid posttranslational modification analysis, identification of components in complex mixtures, and direct mass analysis of gel‐separated proteins. The mass spectrometric methods referred to in this overview include matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS).
Purification of Monoclonal Antibodies
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractThe uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A‐Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion‐exchange chromatography for the preparation of contaminant‐free antibody.
Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatant
Authors: Cooper Helen M. , Paterson Yvonne 
Published: June 2004

AbstractThis unit describes the isolation of the immunoglobulin G (IgG) fraction (containing antibodies of all specificities) from a complex protein mixture such as antiserum, ascites fluid, or hybridoma supernatant. The basic procedure utilizes saturated ammonium sulfate solution to precipitate the IgG fraction, while an alternate protocol describes fractionation of IgG by chromatography on DEAE‐Affi‐Gel Blue resin.
Introduction to Peptide Synthesis
Authors: Fields Gregg B. 
Published: June 2004

AbstractThis overview presents a review of classical and current solid‐phase peptide synthesis techniques. Peptide synthesis has become a more practical part of present‐day scientific research following the advent of solid‐phase techniques, and has practical commercial and scientific implications. This unit provides information on solid‐phase peptide‐synthesis methodology, offering descriptions of different supports, coupling reagents, and practical approaches to the synthesis of modified residues and structures. It also presents information concerning protein synthesis and the purification and analysis of synthetic peptides. This methodology has great implications for the design of synthetic peptides to serve in antibody production.
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 procedure for selecting the antigenic sequence is relatively straightforward. This unit identifies ways to determine the best sequence and to chemically couple the synthetic peptide to a carrier protein to boost the immune response. Another method entails direct synthesis of a MAP covalent multimer of the simple peptide sequence. Peptides can also be coupled to carrier proteins through either their amino or carboxyl groups. Alternate protocols in this unit cover these scenarios. In addition, methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides are provided. Finally, once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis, and a procedure for this is also provided.
Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis
Authors: Buratowski Stephen , Chodosh Lewis A. 
Published: June 2004

AbstractThe DNA‐binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence‐specific DNA‐binding proteins. Proteins that bind specifically to an end‐labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein‐DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA‐binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.
Methylation and Uracil Interference Assays for Analysis of Protein‐DNA Interactions
Authors: Baldwin Albert S. , Oettinger Marjorie , Struhl Kevin 
Published: June 2004

AbstractInterference assays identify specific residues in the DNA binding site that, when modified, interfere with binding of the protein. The protocols use end‐labeled DNA probes that are modified at an average of one site per molecule of probe. These probes are incubated with the protein of interest, and protein‐DNA complexes are separated from free probe by the mobility shift assay. A DNA probe that is modified at a position that interferes with binding will not be retarded in this assay; thus, the specific protein‐DNA complex is depleted for DNA that contains modifications on bases important for binding. After gel purification, the bound and unbound DNA are specifically cleaved at the modified residues and the resulting products analyzed by electrophoresis on polyacrylamide sequencing gels and autoradiography. In the methylation interference protocol presented here, probes are generated by methylating guanines (at the N‐7 position) and adenines (at the N‐3 position) with DMS; these methylated bases are cleaved specifically by piperidine. In the uracil interference protocol, probes are generated by PCR amplification in the presence of a mixture of TTP and dUTP, thereby producing products in which thymine residues are replaced by deoxyuracil residues (which contains hydrogen in place of the thymine 5‐methyl group). Uracil bases are specifically cleaved by uracil‐N‐glycosylase to generate apyrimidinic sites that are susceptible to piperidine. These procedures provide complementary information about the nucleotides involved in protein‐DNA interactions.
UV Crosslinking of Proteins to Nucleic Acids
Authors: Chodosh Lewis A. 
Published: June 2004

AbstractIrradiation of protein‐nucleic acid complexes with ultraviolet light causes covalent bonds to form between the nucleic acid and proteins that are in close contact with the nucleic acid. Thus, UV crosslinking may be used to selectively label DNA‐binding proteins based on their specific interaction with a DNA recognition site. As a consequence of label transfer, the molecular weight of a DNA‐binding protein in a crude mixture can be rapidly and reliably determined. This unit provides 3 protocols for executing DNA‐protein crosslinking; one uses the halogenated thymidine analog bromodeoxyuridine (BrdU) to produce a DNA probe that is especially sensitive to UV‐induced crosslinking. An alternate protocol describes crosslinking using a non‐BrdU substituted probe, and another alternate protocol provides a method for in situ crosslinking.
Determination of Protein‐DNA Sequence Specificity by PCR‐Assisted Binding‐Site Selection
Authors: Pollock Roy M. 
Published: June 2004

AbstractBinding‐site selection is used to determine the target specificity of a sequence‐specific DNA‐binding protein. In this unit, a pool of random‐sequence oligonucleotides is used as the source of potential binding sites. This pool is incubated with extract containing the DNA‐binding protein of interest and the protein‐DNA complexes are isolated by immunoprecipitation with an antibody specific for the protein under investigation. Unbound oligonucleotides are removed by gentle washing, and bound oligonucleotides are recovered, amplified by the polymerase chain reaction (PCR), and used as input DNA for a further round of binding, recovery, and amplification. After four rounds of selection, progress of the procedure is monitored by mobility shift analysis of the selected oligonucleotide pools. In the , individual binding sites are isolated from the appropriate complex on a mobility shift gel, cloned into plasmids, and examined by sequencing.
Introduction of DNA into Yeast Cells
Authors: Becker Daniel M. , Lundblad Victoria 
Published: June 2004

AbstractThe most commonly used yeast transformation protocol is the lithium acetate procedure (described here). It is reasonably fast and provides a transformation efficiency of 105 to 106 transformants/μg. This efficiency rivals that achieved for most, but not all, strains with the more difficult and time‐consuming spheroplast procedure presented here. However, the fastest and easiest of the transformation methods is electroporation, as described in this unit. For a number of strains, electroporation offers the highest transformation efficiency, and may prove especially useful with limiting quantities of transforming DNA. Unlike the lithium acetate procedure, however, electroporation saturates at low DNA levels, restricting its general utility.
Manipulation of Plasmids from Yeast Cells
Authors: Lundblad Victoria , Zhou Heng 
Published: June 2004

AbstractThis unit describes several procedures for manipulating plasmids in yeast cells. The first is a general method to segregate autonomously replicating plasmids from cells: plasmid‐containing yeast cells are grown in nonselective medium, and colonies lacking the plasmid are identified by replica plating. The second, plasmid shuffling, represents a specialized version of plasmid segregation that is useful for analyzing the function of essential genes and for identifying conditional lethal mutations in essential genes. The third approach, plasmid gap repair, is based on the efficient homologous recombination characteristics of yeast cells. Plasmid gap repair can be be used as a method to incorporate mutagenized DNA fragments into a yeast plasmid, rescue genomic mutations onto plasmids, or map alleles of a given gene.
Detection of Hybridized Probe
Authors: Rogers Melissa 
Published: June 2004

AbstractAutoradiography is used to detect and quantitate radioactive probe hybridized to cytological preparations. Autoradiographic film is used to detect 32P‐ or 35S‐labeled probe, and can be useful in experiments dealing with large organs or tissues. Emulsion autoradiography is required to obtain resolution at the level of a single cell. Both procedures are described here.
Whole‐Mount Histochemical Detection of β‐Galactosidase Activity
Authors: Pereira Fred A. 
Published: June 2004

AbstractThis unit describes fixation and staining for b‐galactosidase activity; it has been successfully used on vertebrate embryos and tissue explants. To help achieve consistent results with larger samples, an alternate protocol details preparation of thick sections. Support protocols are provided for clearing of tissues for better resolution and paraffin‐embedding the whole embryo.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M. 
Published: June 2004

AbstractThis unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.
Introduction to Expression by Fusion Protein Vectors
Authors: Riggs Paul , La Vallie Edward R. , McCoy John M. 
Published: June 2004

AbstractThis overview discusses issues involved with creating and manipulating vectors for expression of fusion proteins. The requirements for efficient translation include a promoter and a start codon, along with the fact that the mRNA encoding the protein to be expressed must contain a ribosome‐binding site that is not blocked by mRNA secondary structure. The level of expression is also affected by codon preferences, and may be affected by the coding sequence in other ways that are not yet well understood. In virtually all cases, these problems can be solved by altering the sequence preceding the start codon, and/or by making changes in the 5′ end of the coding sequence that do not change the protein sequence, taking advantage of the degeneracy of the genetic code. However, it is often quicker to solve these problems by making fusions between genes. In this approach the cloned gene is introduced into an expression vector 3′ to a sequence (carrier sequence) coding for the amino terminus of a highly expressed protein (carrier protein). The carrier sequence provides the necessary signals for good expression, and the expressed fusion protein contains an N‐terminal region encoded by the carrier. The carrier sequence can also code for an entire functional moiety or even for an entire protein that can be exploited in purifying the protein, either with antibodies or with an affinity purification specific for that carrier protein. Alternatively unique physical properties of the carrier protein (e.g., heat stability) can be exploited to allow selective purification of the fusion protein. Often, proteins fused to these carriers can be separated from the bulk of intracellular contaminants by taking advantage of special attributes.
Expression and Purification of lacZ and trpE Fusion Proteins
Authors: Hoey Timothy 
Published: June 2004

AbstractFusion proteins are commonly used as a source of antigen for producing antibodies and in many cases can be useful for biochemical analyses. This unit describes two widely used expression systems for producing large amounts of proteins in E. coli. One system expresses lacZ fusions using the pUR series of vectors and the other expresses trpE fusions using the pATH vectors. The gene of interest is first subcloned into either a pUR or pATH vector in the correct reading frame. The correct transformant is selected, grown, and then induced with either IPTG or IAA. Sonication of cells in the presence of protease inhibitors is used to prepare extracts containing both types of fusion proteins, as well as other types of proteins overexpressed in E. coli. The extracts are checked for the presence of fusion protein on an SDS‐polyacrylamide gel.
Inducible Gene Expression Using an Autoregulatory, Tetracycline‐Controlled System
Authors: Shockett Penny , Schatz David 
Published: June 2004

AbstractThe protocols in this unit describe the transfection of adherent cells and the testing of resultant clones for inducible transactivator or target gene protein expression. Stably transfected fibroblast cell lines expressing transactivator and target gene(s) can be derived by first cotransfecting pTet‐tTAk and a plasmid encoding a selectable marker and obtaining stable lines with inducible transactivator expression. These lines are subsequently stably cotransfected with plasmids encoding the target gene(s) and a second selectable marker. The procedure may also be used to cotransfect pTet‐tTAk with the target gene‐encoding plasmid(s) and a single selectable marker plasmid. A support protocol describes methods to test stably transfected cell lines for inducible gene expression, for transient transfection and induction of tet‐regulated plasmids, and for detection of the tTAk gene in cells (or transgenic mice).
Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase Hybrid System
Authors: Elroy‐Stein Orna , Moss Bernard 
Published: June 2004

AbstractThis unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (pT7). Using liposome‐mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7‐3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large‐scale work, protocols are provided for insertion of the pT7‐regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7‐3 into cells grown in suspension or for direct transfection into OST7‐1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse‐labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.
Overview of the HIV‐1 Lentiviral Vector System
Authors: Ramezani Ali , Hawley Robert G. 
Published: June 2004

AbstractReplication‐defective oncoretroviral vectors have been the most widely used vehicles for gene‐transfer studies because of their capacity to efficiently introduce and stably express transgenes in mammalian cells. A limitation of oncoretroviral vectors is that cell division is required for proviral integration into the host genome. By comparison, lentiviruses such as human immunodeficiency virus type 1 (HIV‐1) have evolved a nuclear‐import machinery that allows them to infect nondividing as well as dividing cells. This unique property has led to the development of lentiviral vectors for gene delivery to a variety of nondividing or slowly dividing cells including neurons and glial cells of the central nervous system and others. This unit is intended to provide an overview of HIV‐1 molecular biology and an introduction to successive generations of HIV‐1‐based lentiviral vectors.