p53 Antibody (PAb 240)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Protocol tips
-Mouse (1:1000) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Publication protocol

Cells were lysed in modified RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 NaCl, 10 mM β‐glycerophosphate, 1% NP‐40, 0.25% sodium deoxycholate, 10 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM EDTA, 1 mM vanadate, 20 μg/ml aprotinin, 20 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Whole‐cell lysates were resolved by SDS–PAGE on 4–15% gradient gels and blotted onto nitrocellulose membranes (Bio‐Rad). Membranes were blocked overnight and then incubated sequentially with primary and either HRP‐conjugated (Pierce) or IRDye‐conjugated secondary antibodies (Li‐Cor). Blots were imaged using the Odyssey Infrared Imaging System (Li‐Cor). Protein levels were quantitated using ImageJ (http://imagej.nih.gov/ij/). Primary antibodies used for Western blot analysis included hexokinase 1 (2024, Cell Signaling Technology), hexokinase 2 (2867, Cell Signaling Technology), p53 (NB200‐103, Novus Biologicals), and enolase 2 (8171, Cell Signaling Technology).

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Manufacturer protocol

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