Recombinant Anti-p21 antibody [EPR362] (ab109520)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Western blotting was performed using standard methods. After being treated with indicated drugs, whole-cell or tumour tissue extracts were prepared by radioimmunoprecipitation assay buffer (150 mM sodium chloride, 50 mM Tris pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM NaF, 1 mM Na3VO4, 5 mM EDTA, 1 mM EGTA, 5 μg ml−1 leupeptin, 1 μg ml−1 pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and protease and phosphatase inhibitor cocktail (Calbiochem). The cytoplasmic and nuclear extracts were prepared as previously described based on the different concentration of NaCl (ref. 54). Lysates were centrifuged at 12,000 r.p.m. for 20 min at 4 °C. Protein concentration of the supernatants was determined using a BCA protein Assay Kit (Thermo Scientific). Equal amounts (30–50 μg) of the proteins were resolved by 8–12% SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Millipore). Membranes were blocked in 5% bovine serum albumin for 1 h at room temperature, and then incubated with specific antibodies for different western blot analyses at 4 °C overnight. The bound primary antibodies were detected by secondary conjugates compatible with infrared detection at 700 and 800 nm, and membranes were scanned using the Odyssey Infrared Imaging System (Odyssey, LI-COR). Representative blots are shown from several experiments. The relative optical density of blotting bands was quantified by Image J software (NIH, Bethesda, MD, USA) and normalized to the control.
The following antibodies were used in these assays, all diluted in Primary Antibody Dilution Buffer (Beyotime Biotechnology,China): antibodies directed against p53 (#9282), Pan-ERK (#9102), Pan-AKT (#9272), Histone H3 (#4499, 1:2,000 dilution), α-tubulin (#2125), Phospho-CDK1 (at Tyr15 site, #4539), Phospho-AKT (at Ser473 site, #9271) and Phospho-ERK (at Thr202/Tyr204 sites,#4376) were obtained from Cell Signaling Technologies (1:1,000 dilution). Antibodies directed against p21 (ab109520, 1:2,000 dilution) and BAX (ab32503, 1: 2,000 dilution) were obtained from Abcam. Anti-β-actin antibody was purchased from Sigma (A5441, 1:10,000 dilution). Secondary antibodies were from LI-COR Biosciences, including IRDye 800CW Goat anti-Mouse IgG (926–32210, 1:10,000 dilution) and IRDye 800CW Goat anti-Rabbit IgG (926–32211, 1:10,000 dilution).

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-p21 antibody [EPR362] (ab109520) below.

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