Journal: Protein Science : A Publication of the Protein Society
Article Title: Steps in reductive activation of the disulfide-generating enzyme Ero1p
Figure Lengend Snippet: Disulfide reduction during catalysis by Ero1p and the de-regulated mutant C150A/C295A. Enzyme at a final concentration of 2 μ M was mixed with 100 μ M Trx red . Aliquots were removed from the reactions at the indicated time points, and disulfide status was preserved by blocking with maleimide reagents. The migration of Ero1p on SDS-PAGE at the various time points was examined and compared with wild-type Ero1p (WT), the indicated Ero1p mutants, and DTT-treated Ero1p (WT reduced). (A) The Trx red oxidation reaction was blocked with AMS and separated by SDS-PAGE under nonreducing conditions. The asterisk (*) indicates a species that migrates slower than Ero1p C90A/C349A when blocked with AMS, but faster than this disulfide mutant when blocked with NEM (compare with panel C). (B) The same experiment as in (A) was performed, but the samples were reduced with DTT after AMS treatment before separation by SDS-PAGE. (C) Reactions were blocked with NEM and applied to the gel under nonreducing conditions.
Article Snippet: For oxidation assays, Trx was reduced with 100 m M DTT for 1 h and then desalted using a PD-10 column (GE Healthcare) equilibrated in 50 m M phosphate buffer, pH 7.5, 65 m M NaCl, 0.5 m M EDTA.
Techniques: Mutagenesis, Concentration Assay, Blocking Assay, Migration, SDS Page, Affinity Magnetic Separation