Fluorescein-conjugated iodoacetamide labeling of NAT1 cysteines

NAT1 (1 μM) was incubated with 10 μM BITC or 10 μM PEITC in PBS for 30 min at 37°C. The reactions were quenched with DTT (5 μM) and further incubated with fluorescein-conjugated iodoacetamide (20 μM) for 10 min at 37°C (in the dark). Aliquots (corresponding to 1 mg NAT1) were also spotted on nitrocellulose membranes using a dot-blot apparatus (Bio-rad). Covalent modification of NAT1 cysteines by fluorescein-conjugated iodoacetamide were assessed by fluorescence (Excitation: 494 nm, Emission: 512 nm) with a LAS-4000 apparatus (Fujifilm). The membranes were further used for the immuno-detection of NAT1 using polyclonal anti-NAT1 antibody (166–180, Sigma).