bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐Dimensional Gel Electrophoresis
Authors: Harper Sandra , Mozdzanowski Jacek , Speicher David 
Published: June 2004

AbstractTwo‐dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two‐dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS‐PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two‐dimensional gels, and two‐dimensional protein databases.
Protein Detection in Gels Using Fixation
Authors: Echan Lynn A. , Speicher David W. 
Published: June 2004

AbstractThis unit has been recently updated to include information on preformulated gel stains as well as new protocols for Sypro Ruby and silver staining, and gel imaging methodology. Other previously published protocols are also provided for both rapid and acid‐based Coomassie blue staining, and alternate methods for silver staining (i.e., nonammoniacal silver staining, rapid silver staining, and an enhanced‐background, two‐stage method).
Protein Detection in Gels Without Fixation
Authors: Ursitti Jeanine A. , DeSilva Tara M. , Speicher David W. 
Published: June 2004

AbstractFixatives remove SDS from gels, causing the protein to become entwined in the gel matrix; subsequent extraction from the gel is usually not practical without partial cleavage of the protein or dissolution of the gel matrix. In this unit, methods for the detection of proteins without fixation are described. In general, the sensitivity of the methods is lower than that of protein detection with fixation. Sensitivity is dependent on the volume of the protein band within the gel; hence, sensitivity is the highest for sharp, narrow bands. Techniques described in this unit include protein detection in gels by SDS precipitation, preparation of contact blots, and use of the fluorescent labels IAEDANS and fluorescamine to detect proteins in gels. Several additional methods, including the use of tryptophan fluorescence, guide strips, and minimal protein staining, are discussed in the Commentary.
Capillary Electrophoresis of Proteins and Peptides
Authors: Burgi Dean , Smith Alan J. 
Published: June 2004

AbstractCapillary electrophoresis (CE) is a high‐resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides based on charge‐to‐mass ratios. Separation of proteins based on their isoelectric points is described along with a protocol for optimizing the separation conditions for a given protein. CE is also used for separations of peptides on an analytical scale and as a micropreparative technique‐‐with either multiple separations that are pooled or a single, larger‐scale separation‐‐for the isolation of peptides from a protease digestion. In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.
Immunoblot Detection
Authors: Gallagher Sean 
Published: June 2004

AbstractImmunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody‐antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti‐immunoglobulin‐G (anti‐IgG) antibody (e.g., goat anti‐rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin‐biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.
Enzymatic Digestion of Proteins on PVDF Membranes
Authors: Fernandez Joseph , Mische Sheenah M. 
Published: June 2004

AbstractEnzymatic digestion of membrane‐bound proteins is one of the most widely used procedures for determining the internal amino acid sequence of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Because the final step of protein purification is usually SDS‐PAGE, electroblotting to either polyvinylidene difluoride (PVDF) or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (e.g., SDS or acrylamide) with a high yield. As described in this unit, PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino‐terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF membranes is higher than from nitrocellulose, particularly from higher‐retention PVDF (e.g., ProBlott, Transblot, Westran, or Immobilon Psp). Finally, PVDF‐bound protein can be stored dry, as opposed to nitrocellulose‐bound protein, which must remain wet during handling and storage to prevent loss of peptides during digestion.
Reversed‐Phase Isolation of Peptides
Authors: Henzel William J. , Stults John T. 
Published: June 2004

AbstractReversed‐phase high‐performance liquid chromatography (HPLC) is a fundamental tool for the isolation and analysis of peptides. Peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. This unit presents protocol for separation of 5‐ to 500‐pmol of peptides on a narrow‐bore (2‐mm‐i.d.) or microbore (1‐mm‐i.d.) column. Smaller quantities can be separated capillary HPLC columns, as described. Capillary HPLC columns, however, require a gradient flow rate of 3 to 5 ml/min, which most current HPLC pumps cannot attain without modifications. A procedure is therefore provided for constructing a capillary HPLC system using readily available components. HPLC peaks that appear to be symmetrical may actually contain coeluting peptides. Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis are described for analysis of a small portion of an HPLC fraction to determine the number of components present in a small sample. These methods can be utilized to screen fractions prior to automated sequencing.
N‐Terminal Sequence Analysis of Proteins and Peptides
Authors: Reim David F. , Speicher David W. 
Published: June 2004

AbstractAmino‐terminal (N‐terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N‐terminal end. This unit describes the sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Perkin‐Elmer Procise Sequencer. Sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Hewlett‐Packard Model G1005A sequencer is also described. Methods are provided for optimizing separation of PTH amino acid derivatives on Perkin‐Elmer instruments and for increasing the proportion of sample injected onto the PTH analyzer on older Perkin‐Elmer instruments by installing a modified sample loop. The amount of data obtained from a single sequencer run is substantial, and careful interpretation of this data by an experienced scientist familiar with the current operation performance of the instrument used for this analysis is critically important. A discussion of data interpretation is therefore provided. Finally, discussion of optimization of sequencer performance as well as possible solutions to frequently encountered problems is included.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D. 
Published: June 2004

AbstractThis unit describes the use of inhibitors in cultured cells to prevent N‐linked glycosylation of proteins to yield glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of oligosaccharides on specific proteins or intact cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring
[35S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [3H]mannose using TCA precipitation or endo H digestion. A support protocol details a method for concentrating proteins by acetone precipitation.
Detection of Glycophospholipid Anchors on Proteins
Authors: Doering Tamara L. , Englund Paul T. , Hart Gerald W. 
Published: June 2004

AbstractMany eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI‐anchored proteins. First, the detergent‐partitioning behavior of a protein of interest is examined for characteristics of GPI‐linked species. The partitioning of total cellular and isolated proteins with Triton X‐114 is described in this unit, and precondensation of Triton X‐114, which is necessary to remove hydrophilic contaminants before partitioning, is outlined in a. The protein may also be subjected to specific enzymatic or chemical cleavages to release it from its GPI anchor. Phospholipase cleavage (starting with intact cells or membranes, or with isolated protein) is detailed, and chemical cleavage with nitrous acid is also described. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. A protocol for separation of lipid moieties released from proteins is provided and base hydrolysis of proteins is also presented.
Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine
Authors: Zachara Natasha E. , Cole Robert N. , Hart Gerald W. , Gao Yuan 
Published: June 2004

AbstractFirst, a protocol for increasing the stoichiometry of O‐GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O‐GlcNAc‐modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O‐linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O‐GlcNAc modified proteins, including labeling of O‐GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O‐GlcNAc transferase and O‐GlcNAcase activity, respectively.
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractThis unit describes 32PI labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The protocol described is 32PI labeling of adherent or nonadherent (e.g., hematopoietic) cells with subsequent lysis in a detergent buffer. More rigorous lysis conditions to be used for working with proteins that are difficult to solubilize are also described.
Phosphopeptide Mapping and Identification of Phosphorylation Sites
Authors: Meisenhelder Jill , Hunter Tony , van der Geer Peter 
Published: June 2004

AbstractProtein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.
Analysis of Protein Ubiquitination
Authors: Laney Jeffrey D. , Hochstrasser Mark 
Published: June 2004

AbstractAttachment of ubiquitin (Ub) to a protein requires a complex of enzymes that recognize the substrate and promote Ub transfer. Sequence motifs present in these enzymes may indicate that other uncharacterized proteins containing these motifs have a biochemical function of Ub‐protein ligation, and several in vitro methods are described in this unit for determining if a protein has Ub‐transferring activity. They include psmunoblotting of psmunoprecipitated proteins, affinity purification using His‐tagged ubiquitin, assaying for auto‐ubiquitination of E3, and assaying ubiquitination of a model substrate protein. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
Modification of Cysteine
Authors: Crankshaw Mark W. , Grant Gregory A. 
Published: June 2004

AbstractThis unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N‐ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and
N‐(iodoethyl)‐trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4‐vinylpyridine and acrylamide, are described, and a specialized procedure for 4‐vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.
Modification of Amino Groups
Authors: Geoghegan Kieran F. 
Published: June 2004

AbstractThis unit describes group‐specific modifications of amino groups. These reactions remain valid tools for early‐stage evaluation of structure‐function relationships, but are now valued even more for their applications in the preparation of bioconjugates, affinity columns, biosensors, and tagged macromolecules. Protocols are provided here for reaction of amino groups with succinimidyl esters and isothiocyanates. These methods are broadly useful for the stable coupling to proteins of groups with useful, non‐native functional properties. These include biotin for detection or recovery, fluorescent groups for biophysics or cytochemistry, cross‐linking reagents for making bioconjugates, or metal‐chelators that permit proteins to be loaded with radioisotopes for medical imaging or antitumor therapy. These applications require accurate product characterization, which preferably is performed by mass spectrometry, as described in this unit as a support procedure. A protocol employing succinic or acetic anhydrides to change the charge state of protein amino groups is provided here, as is a procedure for reductive alkylation that leaves their charge unaltered but converts primary amines to secondary or tertiary amines.
Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Analysis of Peptides
Authors: Henzel William J. , Stults John T. 
Published: June 2004

AbstractMatrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is one of the most useful techniques for determining the mass of biomolecules, with exceptional capabilities for mass analysis of peptides. Relative to other ionization techniques, it provides high sensitivity and excellent tolerance of salt and other common buffer components. Routine detection limits for peptides are in the subpicomole range. The ions commonly observed are the protonated molecules (M+H+), which makes data analysis relatively easy. This overview discusses instrument configuration and calibration, sample preparation, along with specific approaches for analyzing peptide mixtures, synthetic peptides, and chemical modifications of peptides.
In‐Gel Digestion of Proteins for MALDI‐MS Fingerprint Mapping
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L. 
Published: June 2004

AbstractMass spectrometry (MS) has emerged as a sensitive, versatile, and rapid method for protein identification, following the advent of electrospray ionization mass spectrometry (ESI‐MS) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). The advantages of MALDI‐MS over ESI‐MS include its relatively high tolerance to contamination from biological matrices, its high sensitivity, the relative ease of interpreting spectra from mixtures, and the formation of singly protonated molecular ions for tandem analysis. Peptide fingerprint mass mapping and partial peptide sequencing using post‐source decay and ladder sequencing by MALDI‐MS in combination with algorithms for sequence database interrogation have the potential for identification and structural investigation of proteins. This unit describes in‐gel digestion for peptide mass mapping of picomole to subpicomole quantities of protein derived from Coomassie‐ or silver‐stained polyacrylamide gels. After digestion, the peptides are extracted from the gel and mass analyzed.
De Novo Peptide Sequencing via Manual Interpretation of MS/MS Spectra
Authors: Addona Terri , Clauser Karl 
Published: June 2004

AbstractDatabases of complete genome sequences are rapidly appearing, and a recent trend in interpretation of peptide MS/MS spectra is database matching. A caveat of database matching is that it can only be successful if the database being searched contains the identical amino acid sequence, or one with high homology, to the peptide being investigated. In instances where high‐quality MS/MS spectra are obtained for which there is no corresponding database match (e.g., when the peptide of interest is derived from a novel protein from an organism whose genome is not known, has post‐translationally modified amino acids, or has multiple amino acid substitutions as compared to the homologous sequence from a different species), de novo (or manual) interpretation of the MS/MS spectrum, as described here, is required. Methods are also provided for verification of the results of the de novo analysis through chemical modification and limited manual Edman degradation of the peptide.
Overview of Macromolecular Electron Microscopy: An Essential Tool in Protein Structural Analysis
Authors: Steven Alasdair C. 
Published: June 2004

AbstractThis unit aims to overview the application of NMR spectroscopy to proteins. It is not intended to provide an exhaustive “how to” guide, but rather to give a flavor of both well‐established and emerging NMR techniques used in the elucidation of protein structure. It starts with a brief introduction to the basic principles of NMR and the information provided by this technique, and goes on to discuss the instrumentation involved, spectral assignment methods for small and large proteins, and the utility of other spin active nuclei (e.g., 13C and 15N) to aid assignment of the latter.
Crystallization of Macromolecules
Authors: Messick Troy , Marmorstein Ronen 
Published: June 2004

AbstractX‐ray crystallography has evolved into a very powerful tool to determine the three‐dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X‐ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, macromolecular complexes, and membrane proteins). The preparation and assessment of crystallization trials is then described, along with a protocol for determining whether the crystals obtained are composed of macromolecule or salt. Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described.
Introduction to NMR of Proteins
Authors: Edwards Andrew J. , Reid David 
Published: June 2004

AbstractThis unit aims to overview the application of NMR spectroscopy to proteins. It is not intended to provide an exhaustive “how to” guide, but rather to give a flavor of both well‐established and emerging NMR techniques used in the elucidation of protein structure. It starts with a brief introduction to the basic principles of NMR and the information provided by this technique, and goes on to discuss the instrumentation involved, spectral assignment methods for small and large proteins, and the utility of other spin active nuclei (e.g.,13C and15N) to aid assignment of the latter.
Introduction to Atomic Force Microscopy (AFM) in Biology
Authors: Goldsbury Claire , Scheuring Simon 
Published: June 2004

AbstractThe atomic force microscope has the unique capability of imaging biological samples with molecular resolution in buffer solution. In addition to providing topographical images of surfaces with nanometer‐ to angstrom‐scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated. Importantly, the measurements are made in buffer solutions, allowing biological samples to stay alive within a physiological‐like environment while temporal changes in structure are measured. This overview provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins. The physical principles of the technique and methodological aspects of its practical use and applications are also described
Raman Spectroscopy of Proteins
Authors: Benevides James M. , Overman Stacy A. , Thomas George J. 
Published: June 2004

AbstractA protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side	‐chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three‐dimensional structure, intramolecular dynamics, and intermolecular interactions. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications that address (1) subunit folding and recognition in assembly of the icosahedral capsid of bacteriophage P22, (2) orientations of subunit main chains and side chains in native filamentous viruses, (3) roles of cysteine hydrogen bonding in the folding, assembly, and function of virus structural proteins, and (4) structural determinants of protein/DNA recognition in gene regulatory complexes. Conventional Raman, UV‐resonance Raman, and polarized Raman techniques are surveyed.