bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of the Gateway System for Protein Expression in Multiple Hosts
Authors: Hartley James L.
Published: June 2004

The Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific recombination. It does not necessarily follow, however, that a gene can be cloned once and expressed in all the available host‐vector combinations with uniformly satisfactory results. This is because different organisms have different mechanisms of translating mRNA into protein, and also because choices always have to be made when designing an expression construct, for example, the presence or absence of a stop codon. This unit reviews Gateway cloning, summarizes aspects of protein expression that limit the universality of the use of one clone in many vectors and hosts, and discusses how conflicts between the structure of a Gateway clone of a gene and the rules of protein expression can be minimized or resolved.The Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific.
Preparation of Soluble Proteins fromEscherichia coli
Authors: Wingfield Paul T.
Published: June 2004

Purification of human IL‐1β is used in this unit as an example of the preparation of soluble proteins fromE. coli. Bacteria containing IL‐1β are lysed, and IL‐1 β in the resulting supernatant is purified by anion‐exchange chromatography, salt precipitation and cation‐exchange chromatography, and then concentrated. Finally, the IL‐1 β protein is applied to a gel‐filtration column to separate it from remaining higher‐ and lower‐molecular‐weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state.
Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli
Authors: Palmer Ira , Wingfield Paul T.
Published: June 2004

High‐level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates and this unit describes preparation of washed pellets and solubilization of the protein using guanidine⋅HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT here or further purified by gel filtration in the presence of guanidine⋅HCl as idescribed here. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS‐PAGE.
Expression and Purification of GST Fusion Proteins
Authors: Harper Sandra , Speicher David W.
Published: June 2004

An increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a “tag” that provides the basis for rapid high‐affinity purification. This unit describes the expression and purification of fusion proteins containing the 26‐kDa glutathione‐S‐transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high‐affinity one‐step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag.
Expression and Purification of Thioredoxin Fusion Proteins
Authors: McCoy John , La Ville Edward
Published: June 2004

This unit describes a gene fusion expression system that uses thioredoxin, the product of the Escherichia coli trxA gene, as the fusion partner. The inherent thermal stability of thioredoxin and its susceptibility to quantitative release from the E. coli cytoplasm by osmotic shock can also be exploited as useful tools for thioredoxin fusion protein purification. In this protocol, a fusion of trxA to any desired gene is constructed and the resulting fusion protein is expressed in an appropriate host strain. Additional protocols describe E. coli cell lysis and fractionation, osmotic release of thioredoxin fusion proteins from the E. coli cytoplasm, and heat treatment to purify some thioredoxin fusion proteins.
Overview of the Characterization of Recombinant Proteins
Authors: Denslow Nancy D. , Wingfield Paul T. , Rose Keith
Published: June 2004

This overview provides guidelines for the characterization of recombinantly expressed proteins (e.g., verifying primary structure and appropriate post‐translational modifications), along with methodologies for characterizing the proteins according to size, X‐ray structure, absorbance, biological activity, and subunit structure. A flow chart presents a suggested path for fully characterizing recombinant protein and involves equipment for HPLC, mass spectrometry, circular dichroism, NMR and fluorescence spectroscopy. Also covered are sources and consequences of contamination in protein solutions.
Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy
Authors: Mach Henryk , Middaugh C. Russell , Denslow Nancy
Published: June 2004

Because each protein (gene product) has a unique amino acid sequence, the particular aromatic amino acid content of each protein results in a unique spectrum in the near‐UV (250 to 350 nm) region. The highly specific microenvironment experienced by each aromatic residue in the three‐dimensional protein matrix results in fine shifts in a protein's spectrum. This unit provides protocols for the detection and analysis of UV spectra of recombinant proteins and their peptide fragments. The unique UV spectral properties of proteins can in turn be used to assess their purity. This application is inherent in the use of a diode array detector to monitor the effluent from a high‐performance liquid chromatography (HPLC) column. A protocol using this technique to assess the purity of recombinant proteins is presented.
Determining the Identity and Structure of Recombinant Proteins
Authors: Denslow Nancy D. , Rose Keith , Righetti Pier Giorgio
Published: June 2004

In this unit peptide mapping protocols with separation of the constituent peptides by high‐performance liquid chromatography (HPLC) analysis and by high‐resolution SDS‐PAGE are presented. Peptide mapping is ideally suited for comparative purposes‐‐for example, combined analysis of the recombinant protein and its natural counterpart (or some other well‐characterized standard). This unit also outlines the general strategy used to determine the linkage pattern of a monomeric recombinant protein containing two intramolecular disulfide bonds. The approach is an extension of peptide mapping, where the aim is to isolate and characterize peptides containing only a single disulfide bond. A two‐dimensional electrophoretic method is also described in which the protein isoelectric point is displayed as a function of pH to yield an electrophoretic titration curve. This method is especially useful for checking for deamidation (e.g., of Asn to Asp) in which additional negative charge is introduced into the modified protein.
Transverse Urea‐Gradient Gel Electrophoresis
Authors: Goldenberg David P.
Published: June 2004

Monitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea‐gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea‐gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.
Measuring Protein Thermostability by Differential Scanning Calorimetry
Authors: Makhatadze George I.
Published: June 2004

Differential scanning calorimetry (DSC) the method of choice to study the conformational stability of biological macromolecules and proteins in particular. This unit presents step‐by‐step protocols for DSC, including sample preparation and interpretation of the results in the simplest cases as well as calibration of the apparatus and maintenance of DSC cells. Various general experimental considerations, including possible pitfalls and errors, are also discussed.
Chromatofocusing
Authors: Williams Alan
Published: June 2004

This unit discusses choice of chromatofocusing medium and buffers, determination of the quantity of medium needed, and preparation of CF buffers. A protocol is provided for a typical chromatofocusing experiment, and support protocols describe regeneration of the chromatofocusing column and several methods for separation of the polybuffer from the purified protein.
One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels
Authors: Ploegh Hidde L.
Published: June 2004

This unit describes isoelectric focusing (IEF) of polypeptides in urea‐containing polyacrylamide slab gels as a result of a pH gradient created by ampholytes (small charged organic molecules) in the gel in response to an electric field. Separation on the basis of isoelectric point produces bands of polypeptides that can be electrotransferred to suitable membranes for further analysis, for example, by staining or immunodetection. If samples have been radiolabeled, they can be visualized by fluorography. This unit contains support protocols detailing special conditions for application of denaturing IEF slab gels for electrotransfer and fluorography in which a scintillator is incorporated into the gel after completion of the run to facilitate detection of radioactively labeled polypeptides.
Electroblotting from Polyacrylamide Gels
Authors: Ursitti Jeanine A. , Mozdzanowski Jacek , Speicher David W.
Published: June 2004

This unit contains procedures for electrophoretically transferring proteins onto a variety of membranes including polyvinylidene difluoride (PVDF) and nitrocellulose, and derivatized membranes. The choice of membrane type for electrotransfer is dependent on the ultimate application for the blot membrane. An alternate protocol is provided for electroblotting in semidry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents.
Detection of Proteins on Blot Membranes
Authors: Harper Sandra , Speicher David W.
Published: June 2004

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one‐dimensional or two‐dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains:
amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities.
Autoradiography
Authors: Bundy Daniel C.
Published: June 2004

This overview provides a thorough discussion of the theory and application of autoradiography for determining the spatial distribution of radioisotope‐labeled substances within a membrane or specimen material. The various types of film are discussed along with instructions on their correct handling. Quantitative autoradiography is described as a means of calibrating experimental results. Importantly, specific guidelines are given for pre‐flashing film, along with exposure and processing procedures.
Digital Electrophoresis Analysis
Authors: Medberry Scott , Gallagher Sean
Published: June 2004

Gel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide‐based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single‐gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image‐capture device, and the images can be easily manipulated using image analysis software. This overview presents reasons for digital documentation and analysis, defines some important key terms for imaging, explains the capture process and reviews the devices used for image capture, and provides an introduction to the software and methods used for one‐ and two‐dimensional digital image analysis.
Enzymatic Digestion of Proteins in Solution
Authors: Riviere Lise R. , Tempst Paul
Published: June 2004

Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid‐promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed‐phase HPLC (RP‐HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.
Chemical Cleavage of Proteins on Membranes
Authors: Crimmins Dan L. , Mische Sheenah M. , Denslow Nancy D.
Published: June 2004

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins bound to membranes. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides. In addition, an describes CNBr cleavage of PVDF‐bound protein previously analyzed by Edman degradation. Finally, a discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.
Overview of Glycoconjugate Analysis
Authors: Varki Ajit , Freeze Hudson H. , Manzi Adriana E.
Published: June 2004

Whereas DNA, RNA, and proteins are linear polymers that can usually be directly sequenced, oligosaccharides show substantially more complexity,having branching and anomeric configurations (α and β linkages). The biosynthesis of oligosaccharides, termed glycosylation, is extremely complex, is not template‐driven, varies among different cell types, and cannot be easily predicted from simple rules. This overview discusses the stereochemistry of mono‐ and oligosaccharides and provides diagrammatic representations of monosaccharides (Fisher projections and Haworth representations) and formulas for representation of oligosaccharide chains. A glossary of terms used in glycobiology is also provided.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit
Published: June 2004

Useful information about glycoconjugates can be obtained by labeling their aglycone (noncarbohydrate) portions‐‐e.g., labeling proteins with radioactive amino acids‐and then using techniques described elsewhere in this chapter to infer the presence, type, and nature of oligosaccharide chains. This unit describes metabolic labeling techniques that provide more specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Following metabolic labeling, the radioactive glycoconjugate of interest is isolated, individual glycosylation sites are identified and separated if necessary, and the labeled oligosaccharides are subjected to structural analysis.
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 presents a group of techniques for analysis of the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. It has been revised from a previous version to include endoglycosidase H, D, F2, and F3 digestions, as well as peptide:N‐glycosidase F (PNGase F), neuraminidase, endo‐β‐galactosidase, and endo‐α‐N‐acetylgalactosaminidase digestions.
Determining the Structure of Oligosaccharides N‐ and O‐Linked to Glycoproteins
Authors: Rudd Pauline M. , Dwek Raymond A.
Published: June 2004

Many proteins involved in biological events are glycosylated. A glycoprotein consists of a mixture of glycosylation variants of a single polypeptide chain, known as glycoforms. It has become clear that a detailed understanding of the roles which glycosylation plays in the biosynthesis, transport, biological function, and degradation of a glycoprotein can only be achieved when the protein and sugar(s) are viewed as an entity. Many glycoproteins can now be modeled by combining glycan sequencing data and oligosaccharide structural information with protein structural data. Pivotal to this approach is sensitive, state‐of‐the‐art oligosaccharide sequencing technology which can give a rapid insight into the glycosylation of a glycoprotein without the need for sophisticated equipment and expertise. This unit gives a detailed introduction into the analysis of glycans, and the many figures will help the user identify which type of experiment needs to be undertaken. Methods for releasing glycans from glycoproteins are followed by protocols for labeling and purifying (by HPLC) the glycans from the rest of the components. Strategies for N‐ and O‐glycan analysis are also included.
Determining the Structure of Glycan Moieties by Mass Spectrometry
Authors: Harvey David J. , Dwek Raymond A. , Rudd Pauline M.
Published: June 2004

This unit complements UNIThere, and describes how to purify samples obtained in that unit for analysis by MALDI‐MS, FAB, and ESI‐MS.
Overview of Protein Phosphorylation
Authors: Sefton Bartholomew M. , Shenolikar Shirish
Published: June 2004

This overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed. The various sites of protein phosphorylation are described along with the roles of the many kinases and phosphatases that regulate phosphorylation. Methods for detecting unlabeled phosphoamino acids, including high‐voltage electrophoresis on thin‐layer cellulose acetate plates, gel‐shift assays, and the use of anti‐phosphopeptide antibodies.
Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies
Authors: Sun Tong , Arlinghaus Ralph B.
Published: June 2004

This unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence‐specific anti‐phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non‐purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence‐specific anti‐phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross‐reactivity of such antibodies, which is a common problem with anti‐peptide antibodies, is also discussed.
Assays of Protein Kinases Using Exogenous Substrates
Authors: Carter Nigel
Published: June 2004

In studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities‐‐cyclic nucleotide‐dependent kinases, protein kinase C and isoforms, casein kinases, Ca2+/calmodulin‐dependent kinases, and tyrosine kinase. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases. In‐gel assays for specific kinase activity are also described. Support protocols describe production of crude cell lysates as a source of enzyme activity, and methods of evaluating assays, including trichloroacetic acid (TCA) precipitation to measure incorporation of radioactivity, adsorption of the labeled material onto P81 phosphocellulose paper, or electrophoretic separation and autoradiography.
Use of Protein Phosphatase Inhibitors
Authors: Weiser Douglas C. , Shenolikar Shirish
Published: June 2004

Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin‐LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.
Analysis of Protein Prenylation and Carboxyl‐Methylation
Authors: Magee Anthony I.
Published: June 2004

This unit describes methods for analysis of prenylation and the carboxyl‐methylation that often accompanies it. The two prenoid groups that have been found attached to proteins‐‐farnesyl (C15) and geranylgeranyl (C20)‐‐are both derived from intermediates in the isoprenoid biosynthetic pathway that utilizes mevalonic acid. In the protocols described here, radiolabeled mevalonate is used to label these intermediates in either intact cells or in vitro; the labeled intermediates then become incorporated into proteins. Alternatively, the preformed radioactive prenyl diphosphates can be used for in vitro translations, as described here. Carboxyl‐methylation of C‐terminal prenylated cysteine residues often occurs subsequent to prenylation. Methods are given for radiolabeling of the methyl group with [3H‐methyl]methionine, that is converted intracellularly intoS‐adenosylmethionine, and for radiolabeling with preformedS‐adenosyl[3H]methionine.
Sample Preparation for MALDI Mass Analysis of Peptides and Proteins
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L.
Published: June 2004

Sample preparation may be the most crucial step in mass spectrometric analysis of peptides and proteins by MALDI. The free dipolar ionic peptides or proteins must be incorporated into a crystalline solid solution that is comprised of excess solvent (the matrix), which modulates desorption/ionization by preferentially absorbing the laser energy at the irradiation wavelength and laser pulse intensity being employed. Usually the cocrystalline sample and matrix are prepared by mixing solutions of each component and permitting the mixture to crystallize through evaporation of the solvents. Two methods employing this approach are presented in this unit:
the dried drop method and the rapid evaporation method.
Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Fit
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L.
Published: June 2004

Peptide fingerprint mass mapping is widely used for initial identification of proteins separated by gel electrophoresis. This method involves the enzymatic in‐gel digestion of proteins to generate peptides, followed by mass measurement of the cleaved peptides. The experimental mass values are then compared with theoretical values from protein databases, calculated using the cleavage specificity of the enzyme. This unit describes the use of the search program MS‐Fit to identify the closest match or matches.
Enzymatic Approaches for Obtaining Amino Acid Sequence: On‐Target Ladder Sequencing
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L.
Published: June 2004

Peptide sequencing by mass spectrometry (MS) is usually based on detecting mass differences associated with various amino acids in the polymer chain. Post‐source decay (PSD) and MS/MS spectra may yield internal peptide sequences. However, determination of the order of the first two, and sometimes the last few, amino acids in the peptide is often problematic without additional experiments. Several enzymatic approaches have proven useful for identifying the N‐ and C‐terminal residues. They involve the use of carboxypeptidases and aminopeptidases to produce peptide ladders for rapid analysis by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). This unit describes the ladder sequence method to generate amino acid sequence information from low‐ to subpicomole quantities of peptides. It can be performed directly on the sample stage, thus minimizing potential sample losses to vials and through sample transfer.
Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using a Nanospray Interface
Authors: Lee Terry D. , Moore Roger E. , Young Mary K.
Published: June 2004

Electrospray (ES) is a concentration‐dependent phenomenon, and operation at high flow rates results in a corresponding high rate of sample consumption. Sensitivity increases are most readily achieved by decreasing the flow rate of sample solution into the electrospray source. In recent years, a number of methodologies have been developed to achieve ES analyses at sub‐ml/min flow rates. There are a large number of microscale ES devices currently in use, but this unit describes in detail a micro‐scale interface for direct liquid introduction without separation.
Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching
Authors: Moore Roger E. , Young Mary K. , Lee Terry D.
Published: June 2004

A procedure is described for rapid, sensitive protein identification utilizing liquid chromatography‐‐ tandem mass spectrometry. The analysis is performed on mixtures of peptides obtained by enzyme digestion. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences.
Introduction to Peptide Synthesis
Authors: Fields Gregg B.
Published: June 2004

A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar‐substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.
Native Chemical Ligation of Polypeptides
Authors: Camarero Julio A. , Muir Tom W.
Published: June 2004

The total synthesis and semisynthesis of proteins allows the site‐specific incorporation of unnatural amino acids, post‐translational modifications, and biophysical/biochemical probes into the target molecule. Among the various chemical and enzymatic approaches available for the synthesis/semisynthesis of proteins, the native chemical ligation technique has proven especially useful and is the exclusive focus of this unit. This unit first discusses how to choose the ligation site(s) in the target protein and then outlines how to obtain the necessary polypeptide building blocks using either chemical synthesis or recombinant DNA expression. Next, the synthesis of a protein by native chemical ligation of two polypeptide fragments is described. The synthesis of a protein from three polypeptide fragments using a sequential native chemical ligation strategy is also described. Support protocols describe how to obtain the necessary polypeptide fragments using either chemical synthesis or recombinant DNA expression.
Detection of Protein‐Protein Interactions by Coprecipitation
Authors: Elion Elaine A.
Published: June 2004

Co‐precipitation of proteins from whole cell extracts is a valuable approach to test for physical interactions between proteins of interest. It may be the method of choice, or may be used in conjunction with other procedures to detect protein‐protein interactions, such as two‐hybrid analyis and co‐purification schemes, and tests of physical associations using purified proteins.
Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Authors: Wouters Fred S. , Bastiaens Philippe I.H.
Published: June 2004

Detection of specific protein‐protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein‐protein interactions from the results of these tests. Fluoresence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.
Identification of Protein Interactions by Far Western Analysis
Authors: Edmondson Diane G. , Dent Sharon Y. R.
Published: June 2004

This unit describes far western blotting, a method of identifying protein‐protein interactions. In a far western blot, one protein of interest is immobilized on a solid support membrane, then probed with a non‐antibody protein. As described, far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways. A protocol is also provided for determining the effects of specific peptide residues or post‐translational modifications on protein‐protein interactions. Many different detection techniques, either radioactive or nonradioactive, can be used. For example, the protein probe may be detected indirectly with an antibody, rather than being labeled radioactively.
Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions
Authors: Cook Neil , Harris Alison , Hopkins Alison , Hughes Kelvin
Published: June 2004

Scintillation proximity assay (SPA) is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps. In SPA, scintillant is incorporated into small fluomicrospheres. These microspheres or “beads” are constructed in such a way as to bind specific molecules. If a radioactive molecule is bound to the bead, it is brought into close enough proximity that it can stimulate the scintillant contained within to emit light. Otherwise, the unbound radioactivity is too distant, the energy released is dissipated before reaching the bead, and these disintegrations are not detected. In this unit, the application of SPA technology to measuring protein‐protein interactions, Src Homology 2 (SH2) and 3 (SH3) domain binding to specific peptide sequences, and receptor‐ligand interactions are described. Three other protocols discuss the application of SPA technology to cell‐adhesion‐molecule interactions, protein‐DNA interactions, and radioimmunoassays. In addition, protocols are given for preparation of SK‐N‐MC cells and cell membranes.
Analytical Centrifugation: Equilibrium Approach
Authors: Laue Tom
Published: June 2004

The specific interaction of biological molecules with one another is fundamental to the biochemistry of all living things. Equilibrium sedimentation is a classic method of biochemistry that provides first‐principle thermodynamic information about the molar mass, association energy, association stoichiometry, and thermodynamic nonideality of molecules in solution. It is one of the most rigorous, powerful, and readily adapted methods for characterizing solution interactions.
Analytical Ultracentrifugation: Sedimentation Velocity Analysis
Authors: Stafford Walter
Published: June 2004

The analytical ultracentrifuge is a high speed centrifuge with an optical system allowing observation of the concentration of macromolecules as a function of radius and time. In sedimentation velocity experiments,relatively high speeds are used so that a boundary is formed between the solution of sedimenting macromolecule and the buffer in which it is dissolved. Analysis of the rate boundary movement and evolution of its shape can yield information about the molar masses of species present as well as stoichiometries and equilibrium constants for their interactions. This overview discusses crucial issues pertaining to sedimentation velocity experiments, including non‐interacting and interacting systems, ideality and non‐ideality, and reversible versus kinetically limited equilibrium scenarios.
Overview of Pepsin‐like Aspartic Peptidases
Authors: Dunn Ben M.
Published: June 2004

The aspartic peptidase family of enzymes has been implicated in a variety of disease states, from stomach ulcers, to breast cancer, and even Alzheimer's Disease. This unit describes the major characteristics of the aspartic peptidases, including mechanism of action, subcellular and tissue localization, and biological substrate specificity.
Caspases
Authors: Denault Jean‐Bernard , Salvesen Guy S.
Published: June 2004

Caspases are a family of cysteine proteases with a strict specificity for aspartate residues involved in inflammatory process and programmed cell death. This overview unit provides basic information on their structure, enzymatic activity, substrate specificity, activation,inhibition and their implication in pathologies. It is intended to be an overview for investigators that are unfamiliar with this family of enzymes but it is also applicable to scientists pursuing research in this field.
Over‐Expression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies
Authors: Parry Marina A. A.
Published: June 2004

This unit describes the over‐expression and purification of active serine proteases and their variants from E. coli inclusion bodies. The strategy includes the folding and purification of a stable zymogen precursor protein, and its later activation with the appropriate convertase to the less stable but active protease. A test to follow the presence of activity in the samples, together with an active‐site titration protocol to determine the number of active sites per mole of total protein are provided. It should be emphasized that although most of the protocols described are applied to a specific example, they are fairly representative of the methods and approaches generally used for laboratory‐scale preparation of other recombinant serine proteases. The critical steps and how this template protocol can be adapted for the purification of other serine proteases are described.
Expression, Purification, and Characterization of Caspases
Authors: Denault Jean‐Bernard , Salvesen Guy S.
Published: June 2004

This unit describes a protocol to obtain milligram amounts of enzymatically active pure recombinant caspases. Specific details for the expression, purification of caspase‐3, ‐6, ‐7, ‐8, ‐9 and ‐10 are discussed along with strategies to obtain particular forms (e.g., the zymogen) of some of them.
Zymography of Metalloproteinases
Authors: Troeberg Linda , Nagase Hideaki
Published: June 2004

Zymography is an electrophoretic technique enabling visualization of the number and approximate size of peptidases in a sample on the basis of their hydrolysis of a protein substrate within the gel. The technique is particularly useful for analyzing the peptidase composition of complex biological samples because visualization depends directly on proteolytic activity. This unit presents a representative zymography protocol for the study of matrix metallopeptidases (MMPs).
Protein Profiling Using Two‐Dimensional Difference Gel Electrophoresis (2‐D DIGE)
Authors: Lilley Kathryn S.
Published: June 2004

2D‐DIGE relies on pre‐electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one gel. The dye‐labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with spot matching between gels. Image analysis programs can then be used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable expression differences to be identified and quantified. This unit describes the DIGE procedure in terms of sample preparation from various types of cells, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.
Laser Capture Microdissection for Proteome Analysis
Authors: Craven Rachel A. , Banks Rosamonde E.
Published: June 2004

Laser capture microdissection is being employed increasingly to isolate specific cell types from tissues, thus overcoming problems of experimental interpretation due to tissue heterogeneity of samples. This unit describes protocols which have been optimized to allow laser capture microdissection of tissues with minimal effect on protein integrity, and their subsequent analysis by techniques including 2D‐PAGE, immunoblotting and SELDI, and discusses the relative merits of this approach.
Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison
Authors: Chevallet Mireille , Tastet Christophe , Luche Sylvie , Rabilloud Thierry
Published: June 2004

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample‐dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for sample‐preparation optimization. These solubilization procedures take into account the constraints brought by two‐dimensional electrophoresis and are thus well suited for proteomic approaches.
Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry
Authors: Link Andrew J. , Jennings Jennifer L. , Washburn Michael P.
Published: June 2004

Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in‐gel digestions by directly identifying protein mixtures in solution.
Quantitative Protein Analysis Using Proteolytic [18O]Water Labeling
Authors: Reynolds Kristy J. , Fenselau Catherine
Published: June 2004

This unit describes the procedure for proteolytic [18O]water labeling of peptides in order to quantitate relative protein levels for a comparative proteomic experiment.
Use of Protein Folding Reagents
Authors: Wingfield Paul T.
Published: June 2004

The reagents and methods for purification of the denaturants guanidine hydrochloride (guanidine‐HCl) and urea are described. Sulfhydryl reagents (reducing agents) and “oxido‐shuffling” (or oxidative regeneration) systems are also discussed.
Dialysis
Authors: Zumstein Louis
Published: June 2004

This appendix 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.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

The first section of this appendix unit discusses basic principles of sterile technique, while the second section discusses medium preparation. A protocol is then provided for subculturing of a monolayer culture grown in petri dishes or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Importing Biological Materials
Authors: Wolf Paula
Published: June 2004

This overview discusses critical issues regarding importing of restricted biological materials along with criteria for handling these materials. Guidelines for importing non‐restricted biological materials are also covered. Recommendations are given for packaging biological materials for export, and finally, the necessary steps for obtaining an import permit application are outlined.
Media Preparation and Bacteriological Tools
Authors: Elbing Karen L. , Brent Roger
Published: June 2004

This appendix presents recipes for basic bacteriological media.
Growth in Liquid or Solid Media
Authors: Elbing Karen L. , Brent Roger
Published: June 2004

This appendix presents basic procedures for growing overnight (and larger) cultures, monitoring growth, and titering and isolating bacterial cultures by serial dilution. In addition, protocols are provided for isolating single colonies by streaking and spreading a plate. Replica plating and strain storage/retrieval is also detailed.
Preparation of Plasmid DNA
Authors: Engebrecht JoAnne , Brent Roger
Published: June 2004

This appendix presents basic procedures for alkaline lysis minipreps, both in tubes and in microtiter plates, and also provides an for boiling lysis. A describes storage of plasmid DNA.
Introduction of Plasmid DNA into Cells
Authors: Seidman Christine E. , Struhl Kevin
Published: June 2004

This appendix presents a procedure for transformation using calcium chloride, and also an alternate procedure for one‐step preparation and transformation of competent cells.
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David
Published: June 2004

This unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. Phenol extraction with ethanol precipitation is described along with an alternate procedure of precipitating DNA with isopropanol. A describes concentration of DNA using butanol.
The Polymerase Chain Reaction
Authors: Kramer Martha F. , Coen Donald M.
Published: June 2004

This appendix describes the methodology behind PCR and gives a method for amplifying DNA enzymatically by the PCR and for optimizing this reaction for the sequence and primer set of interest.
Quantitation of Nucleic Acids with Absorption Spectroscopy
Authors: Gallagher Sean
Published: June 2004

This appendix describes a protocol for measuring absorption of nucleic acid samples at several different wavelengths to assess purity and concentration. A260 measurements are quantitative for relatively pure nucleic acid preparations in microgram quantities. Absorbance readings cannot discriminate between DNA and RNA; however, the ratio of
A at 260 and 280 nm can be used as an indicator of nucleic acid purity.
Theoretical Aspects of the Quantitative Characterization of Ligand Binding
Authors: Sawyer William H. , Winzor Donald J.
Published: June 2004

Living organisms grow, differentiate, reproduce, and respond to their environment via specific and integrated interactions between biomolecules. The investigation of molecular interactions therefore constitutes a major area of biochemical study, occupying a ubiquitous and central position between molecular physiology on the one hand and structural chemistry on the other. While specificity resides in the details of structural recognition, the dynamic interplay between biomolecules is orchestrated precisely by the thermodynamics of the biomolecular equilibria involved. A common set of physicochemical principles applies to all such phenomena, irrespective of whether the interaction of interest involves an enzyme and its substrate or inhibitor, a hormone or growth factor and its receptor, an antibody and its antigen, or, indeed, the binding of effector molecules that modulate these interactions. The binding affinity, binding specificity, number of binding sites per molecule, as well as the enthalpic and entropic contributions to the binding energy are common parameters that assist an understanding of the biochemical outcome. This unit aims to provide an overview of the design and interpretation of binding experiments.
Practical Aspects of Radioligand Binding
Authors: McKinney Michael
Published: June 2004

Radioligand binding assays can be used to characterize receptors in their natural environment (wild type) as well as those transfected into cell lines. They can be used to study receptor dynamics and localization, to identify novel chemical structures that interact with receptors, and to define ligand activity and selectivity. The technique thus serves as a valuable adjunct to other pharmacological and molecular biological approaches. This unit describes the major issues involved in developing a binding assay, such as selecting a radioligand, tissue preparation, optimization of assay parameters, and separation of bound from free ligand. Methods for analysis of the binding data are carefully considered, and tips for troubleshooting a binding assay are also provided.