title: An assay to quantitate reducible cysteines from nanograms of GST-fusion proteins.
abstract: Cysteine residues in proteins are targets of numerous post-translational modifications and play important roles in protein structure and enzymatic function. Consequently, understanding the full biochemistry of proteins depends on determining the oxidation state and availability of the residues to be modified. We have developed a highly sensitive assay that accurately determines the number of unmodified cysteine residues in GST-fusion proteins. Only picomoles of protein are required for each reaction, which are carried out in 96-well glutathione-coated plates. Free unmodified cysteine residues are labeled and quantified using biotin and HRP-conjugated streptavidin. Our assay can be used to quantify reactions targeting sulfhydryl groups in proteins. We demonstrate this assay using full-length and truncation mutants of the SNARE proteins syntaxin1A, SNAP-25B, and synaptobrevin2, which have 0-4 cysteines. We are able to accurately determine the number of cysteine residues in each protein and follow the modification of these cysteines by oxidation and reaction with NEM (N-ethylmaleimide). This assay is as simple as running an ELISA or Western blot and, because of its high resolution, should allow detailed analysis of the chemistry of cysteine residues in proteins.
authors: Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602-5255, USA. dixon_woodbury@byu.edu
authors: Dixon J
authors: DJ
authors: Woodbury
authors: Chris A
authors: CA
authors: Rees
authors: Ammon
authors: A
authors: Thompson
authors: Paul
authors: P
authors: Meiners
authors: April
authors: A
authors: Adams
mesh_terms: D003545:Cysteine / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry*; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D050600:SNARE Proteins / Q000737:chemistry; D019809:Streptavidin
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D019809:Streptavidin; D005982:Glutathione Transferase; D003545:Cysteine; D005033:Ethylmaleimide