Protein a g sepharose beads

The cells or CRC tissues were collected in the IP lysis buffer (Cell Signaling Technology), then the lysates were incubated with IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protein A/G Sepharose beads (Invitrogen, Gaithersburg, MD, USA) at 4 °C for 1 h. The supernatant was collected and incubated with primary antibodies at 4 °C overnight, protein A/G Sepharose beads were added into cell lysates for 4 h of incubation. After washing with IP lysis buffer and PBS for three times, the beads were mixed with loading buffer for western blotting analysis.

Cells were harvested and lysed in whole-cell lysate buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/ml leupeptin, (RIPA buffer, CST, #9806) supplemented with protease inhibitor (1% PMSF). The protein concentrations of the lysates were measured on a spectrophotometer using a Thermo Fisher BCA Protein Assay Kit (23227). The same amount of each whole-cell lysate was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. For immunoprecipitation, 1,000 μg of lysate was incubated with the indicated antibody (1-2 μg) overnight at 4°C followed by incubation for 1H with Protein A /G Sepharose beads (Thermo Fisher 20421). The immunoprecipitates were washed five times with 1×PBS before being resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Quantification of immunoblot band intensity was performed with Image J software.