Fig. 1.

Representative schematic diagram of the biotin switch and the fluorescent switch. Proteins that have been previously S-nitrosylated can be either labeled with biotin-HPDP or an Alexa Fluor-maleimide dye. Step 1 involves blocking of all free thiols with either methyl methanethiosulfonate (MMTS) for the biotin switch or NEM for the fluorescent switch. Step 2 involves the reduction of all S-nitrosylated thiols with ascorbate. Reduced thiols, which were originally S-nitrosylated, are then labeled with biotin-HPDP or an Alexa Fluor-maleimide dye. The Alexa Fluor dye in step 3 of the fluorescent switch can also be 555 or 647 maleimide dyes.