Expression in E.coli and
purification of soluble recombinant proteins. (A)
Purification of ERCC1 on a Talon Co++ charged
resin. Proteins were separated on a 12% SDS–PAGE
gel and stained with Coomassie blue. Lane 1, total cellular proteins;
lane 2, soluble proteins; lane 3, flow through from a Talon column;
lane 4, 10 mM imidazole wash; lane 5, ERCC1 eluted with 500 mM imidazole.
Size markers (kDa) are on the left. (B) Purification
of XPF. Proteins were separated on an 8% SDS–PAGE
gel and stained with Coomassie blue. Sizes (kDa) are shown on the
left. The protein lanes show the high salt eluate from P11 phosphocellulose (P11)
and the Ni++-agarose eluate. (C)
Purification of Δ88ERCC1
on Talon. Proteins were separated on a 12% SDS–PAGE
gel and stained with Coomassie blue. Lane 1, total cellular proteins;
lane 2, soluble proteins, loaded on Talon; lane 3, flow through;
lane 4, 10 mM imidazole wash; lane 5, Δ88ERCC1
eluted with 500 mM imidazole.