bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylation and Uracil Interference Assays for Analysis of Protein‐DNA Interactions
Authors: Baldwin Albert S. , Oettinger Marjorie , Struhl Kevin
Published: June 2004

Interference 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

Irradiation 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

Binding‐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.
Growth and Manipulation of Yeast
Authors: Treco Douglas A. , Winston Fred
Published: June 2004

Preparation of sterile media of consistently high quality is essential for the genetic manipulation of yeast. Recipes for media needed in the protocols in this chapter are provided in this unit. Specific suppliers are recommended for specific ingredients.
Introduction of DNA into Yeast Cells
Authors: Becker Daniel M. , Lundblad Victoria
Published: June 2004

The 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

This 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.
Cryosectioning
Authors: Watkins Simon
Published: June 2004

This unit describes sample preparation and sectioning methods for frozen tissue. Sections of this type are used in a variety of light microscopic procedures including in situ hybridization, immunohistochemistry, and enzyme histochemistry.
Detection of Hybridized Probe
Authors: Rogers Melissa
Published: June 2004

Autoradiography 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

This 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

This 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

This 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

Fusion 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

The 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

This 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

Replication‐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.
Amplification Using CHO Cell Expression Vectors
Authors: Kingston Robert E. , Kaufman Randal J. , Bebbington C.R. , Rolfe M.R.
Published: June 2004

The ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high‐level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.
Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
Authors: Fukuda Minoru
Published: June 2004

This unit describes a collection of methods for chemical labeling of carbohydrates (free oligosaccharides or oligosaccharides conjugated to proteins, peptides, or lipids) by oxidation followed by reduction or by direct reduction. Oligosaccharides can be labeled with either radioisotopes or nonradioactive fluorescent molecules. These labelings allow one to follow the oligosaccharides during chromatography and in cells if labeled by fluorescent molecules. Selective oxidation with mild periodate followed by reduction with tritiated sodium borohydride results in selective radiolabeling of sialic acid residues on oligosaccharides or glycoproteins. Alternatively, treatment of samples with galactose oxidase results in oxidation of galactose or N‐acetylgalactosamine residues at nonreducing termini, rendering these residues suceptible to labeling with NaB[3H]4. Oxidized glycoconjugates can also be labeled using the fluorescent probe lucifer yellow CH. Free oligosaccharides can be labeled by reduction with NaB[3H]4. An additional protocol describes the release and simultaneous labeling of O‐glycan oligosaccharides by alkaline beta‐elimination in the presence of NaB[3H]4.
Sialidases
Authors: Powell Leland D. , Varki Ajit P.
Published: June 2004

Sialic acids are a family of nine‐carbon acidic sugars found at the nonreducing terminus of many glycoconjugates. Sialidases can remove these sugar units selectively from cell surfaces, membranes, or purified glycoconjugates. In this unit, sialidase digestion of purified glycoproteins is described as is treatment of intact cells. The physical properties of the four most useful sialidases are discussed along with their relative activities against sialic acids with different modifications and in different linkages
Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides
Authors: Freeze Hudson H.
Published: June 2004

Carbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar‐chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit offers a group of techniques that will help analyze the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. The sugar chains themselves are not analyzed, but their presence and structure are inferred from gel mobility differences after one or more enzymatic digestions. This approach is most often used in combination with
[35S]Met pulse‐chase metabolic labeling protocols, but they can be applied to any suitably labeled protein (e.g., biotinylated or
125I‐labeled).
Preparation of Glycopeptides
Authors: Powell Leland D.
Published: June 2004

Generation of glycopeptides from glycoproteins is frequently useful when analyzing a protein's oligosaccharide side chains. Freed from the bulk of the polypeptide backbone by proteolysis, glycopeptides can be characterized by a variety of techniques. Extensive proteolysis with pronase or proteinase K results in oligosaccharides with one or a few amino acid residues attached. This technique, detailed in this unit, is often employed as a first step in characterizing oligosaccharides on very large glycoproteins such as proteoglycans and mucins. Limited proteolysis with a specific endoproteinase (e.g., trypsin, a‐chymotrypsin, and V8 protease) is also described, and leaves a larger peptide attached to the oligosaccharide. The resulting glycopeptides are generally suitable substrates for Peptide:N‐glycosidase F, an enzyme useful in defining oligosaccharide‐peptide linkages. Additionally, they can be separated by reversed‐phase chromatography, resulting in a glycopeptide map that is analogous to a peptide map, and used for detection of glycosylation sites.
Composition of Labeled Monosaccharides from Glycosaminoglycans
Authors: Conrad H. Edward
Published: June 2004

Proteoglycans can be metabolically labeled with [3H]glucosamine to incorporate label into glucosamine and galactosamine. Glycosaminoglycans can be released from the proteoglycans and the distribution of labeled glucosamine and galactosamine in the isolated glycosaminoglycans can be determined. A series of acid treatments yields a mixture of disaccharides (uronosyl‐anhydro sugars), free uronic acids, and free anhydro sugars, which are then reduced and separated by paper chromatography. The amount of each labeled component is quantified by scintillation counting. This procedure can also be used to determine the composition of glycosaminoglycans that have not been labeled metabolically. Samples of unlabeled glycosaminoglycans can be treated in the same manner and the hydrolysis/deamination mixture can be reduced with NaB3H4 to give stoichiometric 3H labeling of each reducing sugar component of the mixture.
Analysis of Phosphorylation of Unlabeled Proteins
Authors: Sefton Bartholomew M.
Published: June 2004

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti‐phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit provides a protocol for detection of antibodies by 125I‐labeled protein A or enhanced chemiluminescence (ECL). Detection of enzymatic dephosphorylation using a general or phosphoamino acid‐specific phosphatase and subsequent SDS‐PAGE mobility shift (or a change in activity) is also described.
Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Roussel Robert R. , Stern David F.
Published: June 2004

It is possible to produce anti‐phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross‐react with either the cognate unphosphorylated protein or other phosphoproteins. Unlike conventional antibodies, anti‐phosphopeptide antibodies provide information regarding not only the abundance of a protein but also its activity. Also, unlike general anti‐phosphoamino acid (e.g., anti‐phosphotyrosine) antibodies, which have broad reactivity, anti‐phosphopeptide antibodies may have unique specificity toward the cognate proteins. Such reagents not only facilitate conventional in vitro analysis of phosphoproteins, but also allow heretofore impossible applications, e.g., differential isolation of species of a particular protein that have been phosphorylated at individual phosphorylation sites, as well as analysis of the functional state of a protein in situ by immunohistochemical techniques. This unit provides protocols for the production of both polyclonal and monocloncal anti‐phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to the affinity matrix (Affi‐Gel 10); BSA‐agarose affinity matrix is commercially available.
Sequence Similarity Searching Using theBLASTFamily of Programs
Authors: Wolfsberg Tyra G. , Madden Thomas L.
Published: June 2004

Database sequence similarity searching is carried out thousands of times each day by researchers worldwide and has become a very valuable tool. Over the years, a number of algorithms have been implemented to facilitate database searching. The BLAST (Basic Local Alignment Research Tool) family of sequence similarity search programs allows searches to be done quickly and easily, but with sensitive, yet rigorous statistical expectations. In this unit, which is a completely new version of its predecessor of the same title, the user learns how to access the databases, determine the correct searching strategies, and apply examples of BLAST searches to his or her own data.
Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis
Authors: Ryan Colleen A. , Annunziato Anthony T.
Published: June 2004

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X‐100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.
Chromatin Assembly Using Drosophila Systems
Authors: Fyodorov Dmitry V. , Levenstein Mark E.
Published: June 2004

To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP‐dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.
Overview of Gene Targeting by Homologous Recombination
Authors: Mortensen Richard
Published: June 2004

Formerly UNIT 9.15, this unit has been moved to the opening spot of our new chapter on Embryonic Stem Cell technology. The unit has also been updated, and now includes information about the Cre‐lox and FlP/FRT recombinase systems.
Production of a Subtracted cDNA Library
Authors: Klickstein Lloyd B.
Published: June 2004

For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the λ>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.
AFLP‐Based Transcript Profiling
Authors: Vos Pieter , Stanssens Patrick
Published: June 2004

This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐fingerprinting of double‐stranded cDNA. The protocol described includes the following steps: the isolation of poly(A)+ RNA from total RNA, the synthesis of double‐stranded cDNA, the preparation of template fragments by digestion of the cDNA library with a combination of two restriction enzymes and the ligation of adaptors to the fragment ends, the selective amplification of specific subsets of fragments, and the electrophoretic analysis of these amplification products on standard denaturing polyacrylamide gels. The transcript profiles obtained by this technique are a reliable and efficient tool to identify differentially expressed mRNAs.This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger his unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger
Gene Expression Analysis of a Single or Few Cells
Authors: Klein Christoph A. , Zohlnhöfer Dietlind , Petat‐Dutter Karina , Wendler Nicole
Published: June 2004

The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi‐quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.
Agarose Gel Separation/Isolation of RNA‐Protein Complexes
Authors: Lee Chung‐Sheng Brian , Das Rita , Reed Robin
Published: June 2004

In many methods currently used to analyze RNA‐protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA‐protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA‐protein complexes. Moreover, the use of low‐melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.
Commonly Used Detergents
Authors: Coligan John E.
Published: June 2004

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Commonly Used Reagents and Equipment
Authors: Moore David D.
Published: June 2004

This appendix lists recipes for common reagents and also describes equipment that is typically found in a molecular biology laboratory.

Buffers and Stock Solutions

Acid precipitation solution

Ammonium acetate, 10 M

BBS (BES‐buffered solution), 2×

CaCl2, 1 M

Denhardt solution, 100×

Dithiothreitol (DTT), 1 M

EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)

Ethidium bromide, 10 mg/ml

HBSS (Hanks balanced salt solution)

HCl, 1 M

HeBS (HEPES‐buffered saline) solution, 2×

KCl, 1 M

MgCl2, 1 M

MgSO4, 1 M

MOPS buffer

NaCl, 5 M

NaOH, 10 M

PBS (phosphate‐buffered saline)

Potassium acetate buffer, 0.1 M

Potassium phosphate buffer, 0.1 M

SDS electrophoresis buffer, 5×

SED (standard enzyme diluent)

Sodium acetate, 3 M

Sodium acetate buffer, 0.1 M

Sodium phosphate buffer, 0.1 M

SSC (sodium chloride/sodium citrate), 20×

STE buffer

TAE (Tris/acetate/EDTA) electrophoresis buffer

TBE (Tris/borate/EDTA) electrophoresis buffer

TE (Tris/EDTA) buffer

TEA (triethanolamine) solution

TEN (Tris/EDTA/NaCl) solution

TM buffer, 10×

Tris‐buffered saline (TBS)

Tris⋅Cl [tris(hydroxymethyl)aminomethane], 1 M

TTBS (Tween 20/TBS)
Dialysis and Ultrafiltration
Authors: Zumstein Louis
Published: June 2004

The first section of this unit describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A support protocol describes preparation of membranes for dialysis. The second section describes protocols for ultrafiltration, and the third section describes simple methods of concentrating solutions.
Statistics for the Molecular Biologist: Group Comparisons
Authors: Ryder Elizabeth F. , Robakiewicz Phil
Published: June 2004

In this appendix, some of the statistical tests most commonly used (and misused) in biological research are discussed. These tests are used for comparisons among groups (e.g., t test and ANOVA). A number of other important areas (e.g., linear regression, correlation, and goodness‐of‐fit testing) are not covered. The purpose is to enable the reader to determine rapidly the most appropriate way to analyze data, and to point out some of the most common errors to avoid.
Induction and Measurement of Cytotoxic T Lymphocyte Activity
Authors: Wonderlich John , Shearer Gene , Livingstone Alexandra
Published: June 2004

Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell‐surface antigen recognition and are important in the host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity. The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium‐release assay provided here, labeled antigenic targets are recognized and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.
Blood Collection
Authors: Donovan John , Brown Patricia
Published: June 2004

Blood is most frequently sampled for evaluation of serum antibodies or analysis of surface markers on peripheral blood cells. The protocols offered in this unit describe collection of blood from the orbital sinus or plexus of the mouse, rat, or hamster. With appropriate techniques, small amounts of blood can be obtained with little ill effect on the animal. Collection from the auricular vein or artery of the rabbit is also relatively unstressful to the animal. Bleeding procedures that should be performed on the anesthetized animal include collection from the mouse axillary plexus, cardiac puncture of the mouse, rat, hamster, or rabbit, and collection from the hamster abdominal aorta or vena cava.
Euthanasia
Authors: Donovan John , Brown Patricia
Published: June 2004

Euthanasia of laboratory animals must be performed by trained personnel using appropriate techniques, equipment, and reagents in order to effect a death that is humane and satisfies research requirements. Acceptable methods of euthanasia are painless or minimize distress, and are quick and easy to perform, safe for those performing the procedure, and efficient and economic. They are aesthetically acceptable and are done in the absence of other animals. In addition, these methods do not result in gross histological or histochemical changes that would adversely affect research results. This unit offers protocols for euthanasia employing carbon dioxide asphyxiation, pentobarbital overdose, exsanguination, and cervical dislocation for the mouse, rat, hamster, and rabbit.
Enzyme‐Linked Immunosorbent Assays
Authors: Hornbeck Peter
Published: June 2004

This 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. The can be used to optimize the different ELISAs. The second support protocol presents a method for preparing alkaline phosphatase conjugates.
Purification of Human IgA
Authors: Pack Todd D.
Published: June 2004

This unit describes the purification of human immunoglobulin A (IgA).The main method utilizes an IgA‐binding protein (IgA‐bp) as an affinity reagent, similar to the IgG‐binding proteins, protein A and protein G. An alternate protocol describes a method for isolating IgA1 using affinity column chromatography with Jacalin, an IgA1‐specific lectin. Another alternate protocol describes IgA purification by conventional fractionation using size‐exclusion chromatography.
Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants
Authors: Mowat Allan McI. , Reid George
Published: June 2004

Purified proteins are often poorly immunogenic and in such cases the induction of primary immune responses requires use of an adjuvant. The immune stimulating complex (ISCOM) has a unique ability to provoke a full range of immune response to protein antigens, after both parenteral and oral immunization. This unit describes techniques for incorporating proteins into the ISCOM structure, a process that requires the presence of exposed hydrophobic regions on the protein. The basic protocol outlines a method for preparation of ISCOMs containing inherently nonhydrophobic proteins, to which palmitic acid has been attached covalently. Two alternate protocols are given that do not require covalent modification of the protein. In the first, hydrophobic groups are revealed by acid treatment of the protein. The second describes preparation of ISCOMs containing integral membrane proteins that therefore possess a hydrophobic transmembrane domain.
Cloning, Expression, and Modification of Antibody V Regions
Authors: Morrison Sherie L.
Published: June 2004

Cloned variable (V) regions of antibodies can be expressed joined to any constant (C) region, from either the same or a different species. The resulting antibodies will have the desired associated effector functions. Chimeric antibodies obtained by joining murine V regions to human C regions should have decreased immunogenicity in humans. The process of complementarity determining region (CDR) grafting, in which the CDRs from an antibody of one species are transferred to the framework regions of another species, constitutes a further modification of this approach. The protocols presented in this unit are designed to permit PCR‐based cloning of heavy and light chain V regions. This is an advanced molecular biology protocol and should be employed only by investigators who are sufficiently skilled and experienced.
Depletion of Accessory Cells by Adherence to Sephadex G‐10
Authors: Hathcock Karen S.
Published: June 2004

Accessory cells such as macrophages and other antigen‐presenting cells (APC) in suspensions of mouse spleen and lymph node are removed by means of their preferential adherence to Sephadex G‐10 polymers. Unlike the use of nylon wool columns to remove B and accessory cells (UNIT ), Sephadex G‐10 columns are employed when the main purpose is to yield functional populations of T cells and B cells (by depleting mostly macrophages and other APC). The following technique is simple, fast, and reproducible; however, it results in some retention of B cells on the resin. The basic protocol in this unit involves assembling the column, loading and equilibrating the sterile Sephadex slurry, and passing the cells through the column followed by collection and analysis of eluate. Two support protocols describe preparation of the Sephadex G‐10 slurry for use in the column by fining and sterilization, plus recycling resin from used columns.
Analysis of Flow Cytometry Data
Authors: Sharrow Susan O.
Published: June 2004

This unit provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single‐parameter fluorescence histograms and dual‐parameter contour plots are presented. Next, steps are described for calculating values for signal‐to‐noise ratios when logarithmic amplification is used for data collection. This calculation can be used to compare the amounts of antigenic determinants per cell between different cells stained with the same reagent. Finally, a procedure is described for constructing a calibration curve for logarithmic amplifiers. This calibration curve is required for calculation of signal‐to‐noise values and can also be used to determine if the amplifiers are working properly.
Measurement of Human and Murine Interleukin 2 and Interleukin 4
Authors: Davis Laurie S. , Lipsky Peter E. , Bottomly Kim
Published: June 2004

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL‐2), and interleukin 4 (IL‐4), and of human IL‐2 and IL‐4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The describes the use of the CTLL‐2 line to detect murine IL‐2 and IL‐4 in supernatants. One alternate protocol describes the detection of IL‐2 in samples of human serum or supernatants using CTLL‐2 cells, while other alternate procedures describe the detection and quantitation of murine IL‐4 using a mutagenized subline of CTLL‐2, CT.4S, and the detection of human IL‐4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL‐2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3‐(4,5‐dimenthylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay (). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL‐2, CT.4S, and CT.h4S cell lines.
Measurement of Mouse and Human Interleukin 10
Authors: Mosmann Tim
Published: June 2004

Interleukin 10 (IL‐10) is a multifunctional cytokine produced by T cells and other cell types. It regulates activities of a variety of the cells of the immune system and may play a crucial role in cross‐regulation of different types of T cells. Two methods of quantitating IL‐10 are provided in this unit. The first is an enzyme‐linked immunosorbent assay (ELISA) using a pair of monoclonal antibodies specific for mouse IL‐10. This method has the advantages of high specificity and insensitivity to other cytokines present in test samples. The second method is a bioassay which has the advantages that both human and mouse IL‐10 are detected and that functional protein is measured. Both methods have similar sensitivity.
Measurement of Human and Murine Stem Cell Factor (c‐kit Ligand)
Authors: Smith Kent A. , Zsebo Krisztina M.
Published: June 2004

Stem cell factor (SCF) is an early‐acting, hematopoietic growth factor that binds to the receptor encoded by the proto‐oncogene c‐kit. It is a potent growth factor for primitive bone marrow cells as well as thymocytes. This unit describes three protocols for detecting human and murine SCF. In the first, human or rodent SCF is measured by its ability to stimulate proliferation of the human megakaryoblastic leukemia cell line, UT‐7. Because rat and mouse SCF bind well to human c‐kit, human and rodent SCF can both be measured using the first basic protocol. In an , rodent SCF is assayed by its ability to stimulate proliferation of the clonal murine mast cell line, MC/9. Human SCF is not very active on rodent cells and thus cannot be measured using this protocol. Both of the cell proliferation assays lack specificity because they are capable of detecting other cytokines in addition to SCF. The third protocol is a radioreceptor assay using the human erythroleukemia cell line, OCIM1; it specifically measures murine or human SCF and not other cytokines. Support protocols describe maintenance of UT‐7 and MC/9 cells and preparation of plasma membranes from OCIM1 cells.
Generation of HTLV‐I‐Transformed T Cell Lines
Authors: Nutman Thomas B.
Published: June 2004

Viral transformation of T cells is an effective method for obtaining large numbers of T cells that are easily maintained in the laboratory. Human T cell leukemia/lymphoma virus (HTLV‐I), a type C retrovirus clearly implicated as the etiologic agent of adult T cell leukemia, can both infect and transform normal T cells. This unit presents a protocol in which human T cells are activated and then transformed with HTLV‐I. Detection of virally transformed cells is discussed in the commentary.
Measuring Degranulation of Mast Cells
Authors: Hohman Robert J. , Dreskin Stephen C.
Published: June 2004

This unit describes methods for measuring exocytosis of preformed mediators from secretory granules as an indication of IgE receptor‐mediated activation of mast cells. The first basic protocol describes the measurement of biogenic amines (serotonin and histamine) secreted by activated rodent mast cells (for serotonin) or rodent and human mast cells (for histamine). The second basic and alternate protocols detail techniques for measuring the release of β‐glucuronidase, an enzyme that is synthesized by human and rodent mast cells, stored in secretory granules, and released during degranulation. Methods for assaying other enzymes released during degranulation, such as β‐hexosaminidase and tryptase, are discussed in the . These protocols can also be applied to basophils where appropriate.
Two‐Dimensional Gel Electrophoresis Using the O'Farrell System
Authors: Adams Lonnie D. , Gallagher Sean R.
Published: June 2004

Two‐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 1975. Such equipment is relatively inexpensive and can produce satisfactory results in situations that do not require running large numbers of two‐dimensional gels. Many variations of the original procedure have been described in the literature and by equipment suppliers. The procedures in this unit are appropriate, with minor modifications, for different equipment available from commercial suppliers or custom‐made equipment similar to that described by O'Farrell; detailed instructions are usually supplied by the manufacturer. 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.