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A scheme outlining the method used to detect thiophosphorylated proteins. Step 1. Autophosphorylation reactions are inititated with ATPγS, and quenched with EDTA. Step 2. para-nitrobenzylmeslyate (PNBM) is added to the quenched reactions to alkylate the thiophosphorylated residues. Cross-reactivity occurs with some cysteines. Step 3. An antibody specific for the PNBM derivatized thiophosphate epitope is used to detect proteins that were thiophosphorylated in Step 1.
