PCNA Antibody

Protocol tips

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	-Mouse (1:1000)

Protocol tips
-Mouse (1:1000)

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).

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Manufacturer protocol

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