Purification of wild-type ERp5, ERp5-AGHA, and polyclonal antibodies to ERp5. (A) Purified wild-type (WT) ERp5 (2 μg) and ERp5-AGHA (2 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and stained with coomassie blue dye. (B) Disulfide reductase activity of wild-type ERp5 (100 nM; □) and ERp5-AGHA (100 nM; ○). The relative increase in fluorescence produced by the reduction of di-E-GSSG is reported as a function of time. Di-E-GSSG probe plus DTT alone (×) serves as a negative control. (C) Immunoaffinity-purified anti-ERp5 antibody (0.5 μg/mL) detects wild-type ERp5 (50 ng) but does not detect PDI (500 ng) on western blot analysis. (D) ELISA of immunoaffinity-purified anti-ERp5 antibody (0.1 ng/mL) binding to recombinant His-tagged ERp5, His-tagged ERp72, or His-tagged PDI coated at 0.1 μg per well. Background indicates no bound thiol isomerases. N = 3 in triplicate; error bars represent 2 standard deviations. (E) ELISA of immunoaffinity-purified anti-ERp5 antibody (0.1 ng/mL) binding to recombinant His-tagged ERp5 or His-tagged ERp57. Background indicates no bound thiol isomerases. Proteins were coated at 0.1 μg per well. N = 3 in triplicate; error bars represent 2 standard deviations. These assays were developed with goat anti-rabbit IgG conjugated to HRP and to HRP chromogenic substrate tetramethylbenzidine, and OD was measured at 650 nm (D). In some experiments, the tetramethylbenzidine reaction was terminated with the addition of 50 μL of 0.16 M sulfuric acid, and OD was measured at 450 nm (E). MW, molecular weight; OD, optical density; RFU, relative fluorescence units.