Enhanced chemiluminescence ecl blocking agent

Cells were rinsed with ice-cold PBS and lysed by RIPA lysis buffer with protease and phosphatase inhibitors for 20 min on ice. Then, the cells were centrifuged at 12,000× g for 10 min at 4 °C. Protein extracts (20 μg) were resolved using SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 200 V, 45 min). The protein bands were electrotransferred to nitrocellulose membranes (80 V, 120 min). Membranes were then treated with a 5% enhanced chemiluminescence (ECL) blocking agent (GE Healthcare Bio-Sciences) in saline buffer (T-TBS) containing 0.1% Tween-20, 10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl₂, and 1 mM MgCl₂ at a pH of 7.4 for 1 h, and then incubated with the primary antibody overnight at 4 °C. Subsequently, membranes were washed three times in T-TBS, and proteins were detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by analysis in an ECL plus Western blotting detection system (GE Healthcare Bio-Science).

Cells were rinsed with ice-cold PBS and lysed by RIPA lysis buffer with protease and phosphatase inhibitors for 20 min on ice. Then, the cells were centrifuged at 12,000× g for 10 min at 4 °C. Protein extracts (20 μg) were resolved using SDS polyacrylamide gel electrophoresis (SDS-PAGE; 200 V, 45 min). The protein bands were electrotransferred to nitrocellulose membranes (80 V, 120 min). Membranes were then treated with a 5% enhanced chemiluminescence (ECL) blocking agent (GE Healthcare Bio-Sciences) in saline buffer (TBS-T) containing 0.1% Tween-20, 10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl₂, and 1 mM MgCl₂ at a pH of 7.4 for 1 h, and then incubated with the primary antibody overnight at 4 °C. Subsequently, membranes were washed three times in TBS-T and bound antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by analysis in an ECL plus Western blotting detection system (GE Healthcare Bio-Science) [28 (link)].