Activx desthiobiotin fp serine hydrolase probe

Pancreatic ER fractions as well as total tissue lysates were examined by Western blot as follows: ER fractions (10–20 μg) were separated on Invitrogen Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electrophoresis 4%–20% gradient gels, transferred to nitrocellulose membranes using Bio-Rad (Hercules, CA) TransBlot Turbo rapid transfer system (15 minutes at a constant current of 1.25 A per minigel), and blocked with 5% bovine serum albumin in Tris-buffered saline buffer with 0.075% Tween 20. Subsequent incubations and washes were performed in Tris-buffered saline with 0.075% Tween 20. Primary antibodies used against lipase, carboxyl ester lipase (Cel) (sc-34878), signal recognition particle 72 were from Santa Cruz Biotechnologies (Santa Cruz, CA). Extracellular signal-regulated kinase 1/2 antibody was from Cellular Signaling Technologies (Beverly, MA). ActivX Desthiobiotin-FP Serine Hydrolase Probe and streptavidin–horseradish peroxidase were from Thermo Fisher Scientific. ER fractions (50 μg) were incubated with probe (2 μg/mL) in 10 mmol/L Tris-HCl, pH 7.4 for 20 minutes at room temperature, then Triton X-100 (Bio-Rad) was added to 1% and the clarified mixture was incubated for a further 20 minutes at room temperature. Samples then were prepared by addition of gel loading buffer and further processed for Western blot as described earlier.