Search

INTRODUCTIONThe sites of trypsin cleavage can be restricted when the ε-amino group of lysine is blocked, for example, by succinic anhydride. This can be useful for obtaining overlapping peptides during classical protein sequencing strategies. For example, in selective peptide cleavage with trypsin, it may be desirable to block the ε-amino groups of lysine so that the enzyme attacks only at arginine peptide bonds. Another advantage of succinylation is that the reagent places substantial negative charge on the protein because the positively charged lysine side chain is replaced by a negatively charged carboxyl group of the succinate half-amide. Hence, succinylation may be used to solubilize a protein while blocking its lysine groups (in general, succinylated proteins are soluble at pH7). The attached succinyl moiety forms a stable linkage to most protein treatments, but may be cleaved off with 6 N HCl at 100ºC.

INTRODUCTIONSYPRO Ruby is a sensitive ruthenium-based fluorescent dye that, similar to silver stains, interacts with basic amino acids, including lysine, arginine, and histidine. Recent studies have shown that this dye is as sensitive as silver staining and completely compatible with matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The staining method has only three steps: fixation, staining, and destaining. Since SYPRO Ruby is very expensive, this dye might be limited to micropreparative gels, as the cost to run the large number of gels done in quantitative studies is prohibitive.

INTRODUCTIONReversible masking of ε-amino groups of lysine is a valuable procedure for limiting trypsin hydrolysis of proteins to arginine peptide bonds. Citraconic anhydride is a reversible blocking agent of lysyl side chains. A major advantage of citraconylation is the ease with which the blocking group can be removed under conditions that will not lead to protein denaturation.

INTRODUCTIONThis protocol describes a method for concentrating gel bands or spots excised from 1D or 2D acrylamide gels. Several replicate protein bands (or spots) are often required to provide sufficient material for sequencing. In these situations, it is necessary to excise several stained protein bands or spots from the gels and pre-concentrate them prior to electroblotting for amino-terminal sequencing or internal sequence analysis following in-gel proteolytic (or chemical) fragmentation. In the method described below, concentration is done by re-electrophoresis of the gel pieces in a simple SDS-PAGE gel poured in a single conventional glass Pasteur pipette.

INTRODUCTIONChemical reagents that selectively cleave at tryptophan residues have contributed greatly to the elucidation of protein structure and function. This protocol uses ο-iodosobenzoic acid for specific cleavage at tryptophan residues.

INTRODUCTIONIn this protocol, sample proteins are first separated by either one- or two-dimensional gel electrophoresis. The gel is briefly stained and destained, and the proteins are excised for peptide mapping. Peptide fragmentation (either enzymatically or chemically) is performed in situ in the gel pieces, and the partial digest is separated in a second gel.

INTRODUCTIONThis article provides recommendations for immobilizing proteins in an array format as well as general guidelines for preparation and storage of samples. Other considerations that are discussed include reaction conditions (such as protein concentration and buffer composition) appropriate for the protein(s) being studied and techniques to eliminate common problems with protein microarray printing.

INTRODUCTIONThe zinc/imidazole staining procedure for visualizing proteins in acrylamide gels is based on differential salt binding. Because protein-bound salts (e.g., dodecyl sulfate or the heavy cation zinc) are chemically less active than free zinc ions in the gel, precipitation of an insoluble salt is much slower in those regions of the gel occupied by proteins than in the gel background where zinc dodecyl sulfate precipitates. The result is a “negative stain,” with translucent proteins and an opaque gel background, due to zinc dodecyl sulfate precipitation. The sensitivity of the method has been markedly improved by altering the composition of the precipitated salt to a complex of zinc and imidazole. This protocol provides two methods: reverse stain using imidazole, SDS, and zinc, and double-staining using Coomassie blue stain followed by zinc/imidazole.

INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu+ generated under alkaline conditions at sites of complexes between Cu2+ and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.

INTRODUCTIONThe Cleveland method for peptide mapping of proteins relies on partial hydrolysis of the protein yielding a considerable number of large peptides that can be readily identified by SDS-PAGE or tricine-SDS-PAGE. Small peptides obtained with a total digest are often poorly fixed within the gel and usually contribute nothing to the peptide map. In the method described here, the protein is partially digested in solution, and the resulting peptide fragments are separated by SDS-PAGE.

INTRODUCTIONSelective cleavage of Asn-Gly bonds in proteins by hydroxylamine generates a relatively small number of large peptide fragments. This protocol describes a procedure for performing Asn-Gly bond cleavage in solution.

INTRODUCTIONSelective cleavage of a protein by cyanogen bromide generates a small number of relatively large peptide fragments. This protocol describes protein cleavage with cyanogen bromide, using ~100-fold molar excess of cyanogen bromide over the methionine. The reaction is performed under mild acidic conditions (e.g., 70% formic acid), under nitrogen, and in the dark. All reagents are volatile and can be readily removed by lyophilization.