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. 1999 Apr 27;96(9):4862–4867. doi: 10.1073/pnas.96.9.4862

Figure 2.

Figure 2

The use of NEM as a sulfhydryl blocking agent in protein footprinting by NTCB. The signs across the row denoted AMPPNP specify whether the ATP analog was absent (−) or present (+) during partial cysteine cyanylation by NTCB, and those across the row denoted NEM similarly specify whether excess NEM was added at the time of unfolding the cyanylated protein at pH 9. All proteins were 32P-labeled at their N-terminal HMK tag and, after electrophoresis, the gel was dried for autoradiography. Lane 1, size markers obtained by partial cleavage at the 10 cysteinyl residues of the L125C protein; lane 4, the size markers in lane 1 treated with NEM; lanes 2 and 3, NTCB footprinting of the L125C mutant protein without NEM treatment; lanes 5 and 6, NTCB footprinting of the L125C mutant protein with NEM treatment; lanes 7 and 8, NTCB footprinting of the single Cys-125 mutant with the incorporation of NEM treatment. The amount of NTCB used in the treatment of this single cysteine protein was one-half of that used for the proteins with multiple cysteines.