Anti mouse igg secondary antibody conjugated to hrp horseradish peroxidase

Total protein was extracted using RIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) with cocktail protease inhibitors and PMSF (Roche, Rotkreuz, Switzerland). Equal quantities of proteins were loaded into each lane and separated on polyacrylamide SDS gel. Then the proteins were transferred to the PVDF or nitro cellulose membrane (BIO-RAD Immun-Blot^® Membrane) and the membrane was further incubated and placed on a shaker with blocking buffer containing 10% skimmed milk in TBS for 1 hr at room temperature. Then they were incubated with primary antibody (Supplementary Table S1) for 1 h at 37 °C or overnight in 4 °C with consistent shaking. This was followed by incubation with either anti-rabbit IgG or anti-mouse IgG secondary antibody conjugated to HRP horseradish peroxidase (Cell Signaling, Danvers, MA, USA) in blocking buffer for 1 hr at room temperature. The ECL substrate Chemiluminescence signal was detected using ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Total protein was extracted using RIPA lysis buffer (Cat #9806, Cell Signaling, Danvers, MA, USA) with the addition of 1:100 protease PMSF inhibitors (Cat # 5872S, Cell Signaling, Danvers, MA, USA). When running the Western blot, equal quantities of proteins were loaded into each well and separated by size on a polyacrylamide SDS gel. Then, the proteins were transferred to a PVDF membrane (BIO-RAD Immuno-Blot^® Membrane). Once the transfer was complete, nonspecific binding was blocked by incubating the membrane on a shaker with a blocking buffer comprised of 10% skimmed milk in TBS for 1 h at room temperature. The membranes were then incubated with a primary antibody (Supplementary Table S1) at 4 °C overnight. This was followed by incubation with either anti-rabbit IgG or anti-mouse IgG secondary antibody conjugated to HRP horseradish peroxidase (Cell Signaling, Danvers, MA, USA) in antibody dilution buffer for 1 h at 37 degrees Celsius. Washing steps were performed after primary and secondary antibody incubations, which involved 3 washes with TBS buffer + 1% Tween for 5 min each. The ECL substrate Chemiluminescence signal was detected using the ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Total protein was extracted using RIPA lysis buffer (Cat #9806, Cell Signaling, Danvers, MA, USA) with the addition of 1:100 protease PMSF inhibitors (Cat # 5872S, Cell Signaling, Danvers, MA, USA). Equal amounts of proteins were injected into each well and separated using SDS-PAGE gel electrophoresis before the Western blot was conducted. The proteins were then transferred to the PVDF membrane BIO-RAD Immuno-Blot^® Membrane. Nonspecific binding was stopped once the transfer was finished by shaking the membrane in a blocking buffer made of 10% skimmed milk in Tris-Buffer solution pH 7.4 (TBS) for one hour at room temperature. The membranes were next exposed to a primary antibody for an overnight incubation at 4 °C. Then, for 1 h at 37 °C, either an anti-rabbit IgG or anti-mouse IgG secondary antibody conjugated to HRP horseradish peroxidase (Cell Signaling, Danvers, MA, USA) was added. After primary and secondary antibody incubations, washing processes were carried out. These required three washes with TBS buffer + 1% Tween for 5 min each. The membraned was visualized using ECL substrate Chemiluminescence signal on Bio-Rad Laboratories ChemiDoc imaging equipment (Hercules, CA, USA).