Gel doc 1000 electrophoresis documentation

Human CRC tissues and CRC cell lines SW480 and LoVo were collected, and lysed in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor and phosphatase inhibitor (Roche, Mannheim, Germany). Protein extracts were separated by 6%-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, MA, USA). The membranes were blocked in TBST containing 5% skimmed milk for 2 h, followed by incubation with primary antibodies (1:1000) overnight at 4˚C. Subsequently, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h. After washed with TBST, the interest proteins were detected using enhanced chemiluminescence (Millipore Corporation, MA, USA), followed by exposure on the Gel Doc 1000 Electrophoresis Documentation (Bio-Rad, CA, USA).

Cells with different treatments were collected and washed with ice-cold PBS, then lysed in ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor and phosphatase inhibitor (Roche, Mannheim, Germany). Protein extracts were quantitated by the bicinchoninic acid assay, denatured by boiling, separated by 10% sodium dodecyl sulfate poly-acrylamide gel electrophoresis and blotted onto the PVDF membranes. After blocking with 5% bovine serum albumin (Solarbio, Beijing, China) at room temperature for 2 h in Tris-buffered saline and Tween 20 (TBST), the membranes were probed with the primary antibodies (1:1,000) and incubated at 4°C overnight. Subsequently, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h. After washing with TBST, the proteins were detected using enhanced chemiluminescence (Millipore Corporation), followed by exposure on the Gel Doc 1000 Electrophoresis Documentation (Bio-Rad). Three separate experiments were performed for each group.