Mouse anti human gapdh monoclonal antibody

The homogenized gastric cancer and noncancer samples were lysed in RIPA lysis buffer. The lysate was centrifuged (13800 g) at 4°C for 30 min. The supernatant was used for Western blotting analysis. The aliquots containing approximate 20 μg protein each were denatured with sodium dodecyl sulfate (SDS) loading buffer, resolved by electrophoresis in a 10% SDS polyacrylamide gel, and then transferred onto a polyvinylidene fluoride membrane. After blocking with 5% nonfat milk for 1 h, the membranes were incubated with a rabbit monoclonal antibody against TM4SF1 (1:1000 dilution, Abcam) or mouse anti‐human GAPDH monoclonal antibody (1:2000 dilution, Sigma‐Aldrich) at 4°C overnight. After washing with Tris‐buffered saline with Tween‐20 (TBST) for three times with 10 min each, the membranes were probed with the horseradish perioxidase (HRP)‐conjugated mouse anti‐rabbit IgG antibody (1:2000 dilution, Sigma Chemical Co., St Louis, MO) at room temperature for 1 h. After washing with TBST, the membranes were developed using a chemiluminescence phototope‐horseradish peroxidase kit according to the manufacturer's instructions (Pierce, Rockford, IL). The band intensity was measured by densitometry using Quantity One software (Bio‐Rad, Hercules, CA). The TM4SF1 protein level was normalized to that of GAPDH.