Publication protocol
Cells were prepared by washing with cold PBS and then lysed with 1× lysis buffer (Cell Signaling Technology) for 1 h. The supernatants were collected after centrifugation 12,000 rpm for 10 min at 4 °C, and then incubated with appropriate antibodies for 1–2 h followed by adding 20–30 μl protein G beads (Santa Cruz Biotechnology) at 4 °C overnight. The beads were washed three times with IP buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 1% Nonidet P40), followed by immunoblot (IB) analysis. For the IB assay, cells were harvested and lysed with 1 × RIPA buffer (Cell Signaling Technology), followed by samples were boiled for 5 min together with 2 × or 5 × loading buffer to be used to perform SDS-PAGE. Proteins were further transferred onto 0.22 μm PVDF membrane (Roche). The membrane was sealed with 5% fat-free milk in Tris-buffered saline added 1% Tween-20 (TBS-T) for 1–2 h at room temperature and then incubated with appropriate primary antibody at 4 °C overnight. The membrane was washed three times for 10 min each with TBS-T and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After washing three times with TBS-T, the membrane was flushed with super ECL (KeyGEN, Nanjing, China) or Immobilon Western HRP (Millipore). Then the bands were exposed by X-Ray film (FUJI, JAPAN) or detected by ChemiDoc Touch (Bio-Rad).
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Western blotting PCNA using PCNA Antibody from Proteintech Group. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.