  The influence of thiol blocking on the resolution of basic proteins by
two-dimensional electrophoresis was investigated. Cysteine blocking greatly
increased resolution and decreased streaking, especially in the basic region of
the gels. Two strategies for cysteine blocking were found to be efficient:
classical alkylation with maleimide derivatives and mixed disulfide exchange
with an excess of a low molecular weight disulfide. The effect on resolution
was significant enough to allow correct resolution of basic proteins with
in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic
cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These
results demonstrate that thiol-related problems are not solely responsible for
streaking of basic proteins on two-dimensional gels.
