p53 Antibody (DO-1): sc-126

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Tumor samples collected from CRC patients were flash-frozen in liquid nitrogen and then lysed in ice-cold lysis buffer (20mM Tris-HCl pH 7.8, 150mM NaCl, 2mM EDTA pH 8.0, 1% NP40, 10% glycerol, supplemented with 10 mM NaF, 10 mg/mL Leupeptin, 200mM Na2VO4, 5mM PMSF, and Aprotinin) (Sigma-Aldrich). Homogenization was performed on ice with a POLYTRON® system PT 1200 E (Kinematica). Lysates were centrifuged at 13.000 rpm 4°C for 30 min and the supernatants were collected. Cells were lysed on ice by using RIPA lysis buffer (50mM Tris-HCl pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150mM NaCl, 2mM EDTA, 50Mm NaF) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Gendeport, Barker, TX, USA). The total protein concentration was measured by the BCA protein assay kit (Thermo, Rockford, IL, USA). The protein extractions were denatured by SDS-sample buffer and incubated at 95°C for 10 min. The denatured protein mixture was subjected to SDS-PAGE, subsequently transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), and blocked with 5% non-fat milk in Tris-buffered saline pH 7.6 containing 0.1% Tween-20. Then the membranes were probed with the corresponding primary antibodies overnight at 4°C. After washing with TBS-T buffer for three times (10 min for each wash at room temperature), the membranes were incubated with an anti-rabbit secondary antibody (1:10000, sigma, Cat# 7074) or anti-mouse secondary antibody (1:3000, Cell Signaling Technology, Cat# 7076) for 1 h at room temperature followed by washing with TBST three times (10 mins for each wash at room temperature). The antigen–antibody complexes were detected using West-Q Pico Dura ECL Solution (Gendeport, Barker, TX, USA). The intensities of protein bands on the films were quantified by scanning densitometry using the Image J software (NIH).

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