A Nano-assay To Measure Modification Of Cysteine Residues In GST-fusion Proteins

Abstract

Cysteine residues are the target of numerous posttranslational modifications and play important roles in protein structure and enzymatic function. Because of this, much research on the biochemistry of proteins is dependent on understanding the activity and state of these residues. Many current methods for measuring modified and unmodified cysteine residues in proteins are cumbersome and often lack sensitivity, requiring large amounts of protein. We have developed a highly sensitive and simple assay that accurately measures the relative amounts of free cysteine residues in GST-fusion proteins using 96 well glutathione-coated plates. Free-unmodified cysteines are labeled and visualized using biotin and HRP-conjugated streptavidin. Our assay can be used to quantify the extent of reactions targeting -SH groups in proteins. We demonstrate this assay using full-length and truncation mutants of the SNARE proteins: syntaxin-1A, SNAP-25B and synaptobrevin, which have 0-4 cysteines. With this assay we are able to quantitatively measure the number of cysteine residues modified in reactions such as palmitoylation and oxidation. This assay is as simple as running an ELISA or western and should allow greater elucidation of the chemistry of cysteine residues in proteins cysteine due to its high resolution.