Pierce tmb subs trate kit

Protein from the cell pellet was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fischer scientific, cat No. 23227) 20ug of protein was load in 4–12% Bis-Tris SDS-polyacrylamide gel (Invitrogen). Samples were transferred into a PVDF membrane (Thermo Fisher) and membrane blocking was performed using 2% nonfat dry milk. Primary antibody incubation was overnight and secondary antibody for one hour. The membrane was washed between antibodies incubation using 1X PBS-T three times each 10min. HRP was detected using Pierce™ TMB Subs trate Kit and membrane was exposed to HyBlot CL autoradiography film (Denville Scientific) to visualize the protein band.

PFG and PP cells (1x10⁷ cells) were harvested using 0.25% Trypsin, washed twice with PBS, pelleted and stored at −80c. Protein from cell pellet was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fischer scientific, cat No. 23227). 20ug of protein was loaded onto a 4–12% Bis-Tris SDS-polyacrylamide gel (Invitrogen). Samples were transferred into a PVDF membrane (Thermo Fisher) and membrane blocking was performed using 2% nonfat dry milk. The membrane was stained in primary antibody diluted in 2% nonfat dry milk overnight at 4°C. After washing 3 times with 1X PBS-T for 5 mins each, the membrane was stained in secondary antibody diluted in 2% nonfat dry milk for 1 hour at room temperature. The membrane was washed 3 times with 1X PBS-T for 5 mins each. HRP was detected using Pierce™ TMB Substrate Kit and membrane was exposed to HyBlot CL autoradiography film (Denville Scientific) to visualize the protein band.