2-nitro-5-thiocyanobenzoic acid (NTCB) cysteine footprinting

NTCB cleavage was performed as previously described (Jacobson et al., 1973). Purified proteins were incubated for 1 h at 37°C in reduction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5 mM DTT). Reduced proteins were dialyzed against 50 mM Tris-HCl pH 8.0 and 100 mM NaCl to remove excess DTT. A 10-fold molar excess of NTCB over molar concentration of total cysteines was added to each sample. After incubation of 2 h at 20°C proteins were dialyzed against sample buffer to remove non-reacted NTCB. For denaturation, proteins were buffer exchanged into unfolding buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5 M urea). Cleavage was initiated with a raising of the pH of the reaction to pH 9.0 with 1 M NaOH. The reactions were incubated overnight at 37°C, stopped by addition of 3 mM β-mercaptoethanol and analyzed by SDS-PAGE.