Increasing the platelet releasate pH from 7.4 to 8.6 enhances both latent TGF-β1 activation and thiol labeling. (A,B) Platelet releasate at pH 7.4 was adjusted to pH 8.6 by adding NaOH and incubated for 10 minutes at 37°C. Samples were neutralized to pH 7.4 by adding HCl. TGF-β1 activity was measured using both the ELISA (A) and MLEC assay (B). Error bars represent SD. (C,D) Platelet releasates at pH 7.4 and pH 8.6 were labeled with MPB (100 μM) before (C) or after (D) pH adjustment. Proteins labeled with MPB were detected with streptavidin (SA)–HRP before (non-IP) or after (IP) immunoprecipitation with an antibody to TGF-β1. TGF-β1 was detected with an antibody to TGF-β1. The amount of MPB incorporated into TGF-β1 increased when MPB was added before the pH shift, whereas less labeling occurred when MPB was added after the pH shift.