Rabbit anti human pd l1 antibody

Cell protein was harvested using RIPA buffer (G Biosciences, St. Louis, MO, USA) supplemented with Halt protease inhibitor (Thermo Fisher Scientific, Grand Island, NY, USA). Protein concentration was measured using the DC protein assay (Bio‐Rad, Hercules, CA, USA). Electrophoretic separation of protein (12–20 μg/well) was performed using 4–15% gradient polyacrylamide gels (Bio‐Rad). Separated protein was transferred onto PVDF membranes (Bio‐Rad), which were then blocked for 1 h at room temperature in Tris‐buffered saline containing 0.1% tween (TBS‐T) with 5% fat‐free milk (Bio‐Rad), followed by overnight incubation at 4°C with rabbit anti‐human PD‐L1 antibody (Cell Signaling Technologies, #13684T, Danvers, MA, USA) (1:1000 dilution) or mouse anti‐human β‐actin antibody (1:10 000 dilution) (Cell Signaling Technology, #3700 S) in 5% fat‐free milk with TBS‐T. Membranes were washed in TBS‐T and incubated for 20 min at room temperature with a 1:2000 dilution of horseradish peroxidase‐conjugated goat anti‐rabbit antibody (Promega, #W4011, Madison, WI, USA) or rabbit anti‐mouse antibody (Cell Signaling Technology, #7076 S) in 5% milk with TBS‐T. Protein signals were developed using the WesternBright ECL HRP substrate (Advansta, #K‐12045, San Jose, CA, USA) and measured using a Chemi‐Doc MP scanner (Bio‐Rad).

Cells from cultures were lysed in RIPA buffer (Cell Signal Technology) containing 2 μl/ml protease inhibitor cocktail (Sigma-Aldrich). Protein samples were separated by electrophoresis using pre-casted mini PAGE (Bio-Rad) at 120 V for 1.5 h. The separated proteins were transferred onto PVDF membrane at 100 V for 1 h. The membrane was blocked at room temperature with 5% bovine serum albumin in Tris-Buffered Saline and 0.5% Tween 20 (TBST) buffer for 1 h and washed three times with TBST with each wash being 5 min. The membrane then was incubated overnight with rabbit anti-human PD-L1 antibody (Cell Signal Technology) at 1:500 dilution. After washing three times with TBST, the membrane was incubated for 2 h at room temperature with horseradish peroxidise conjugated goat anti-rabbit antibody (Cell Signal Technology) at dilution 1:2500. The membrane was incubated with ECL for 5 min and imaged by GelDoc UV illuminator (Biorad Laboratories).