Recombinant Anti-Smad4 antibody [EP618Y] (ab40759)

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

Cells (2 × 106 cells/T75 flask) were seeded in the presence of 30 μM MnTE-2-PyP or an equal volume of PBS for 24 h before either sham or 2 Gy irradiation and 24 h later the cells were collected and washed in 1× PBS. Cell pellets were lysed by cell lysis buffer [120 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP-40 and complete protease inhibitor cocktail tablets (1 tablet/50 ml, cat. no. 11697498001; Roche Diagnostics, Indianapolis, IN)]. After incubation for 30 min on ice, cell lysates were sonicated for 8–10 pulses at 80% amplitude. After sonication, lysates were centrifuged at 4°C for 15 min at 11,000g and supernatants were collected. Whole cell lysates were assayed for total protein concentrations with a Bradford reagent (Amresco® Inc., Solon, OH) using bovine serum albumin as a standard. Total proteins (40 μg) were loaded on to Bolt® 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Life Technologies, Grand Island, NY). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween™ 20) for 2 h, the membranes were incubated with primary antibodies overnight. Membranes were then washed with TBST and incubated with a 1:10,000 dilution of HRP-conjugated secondary antibodies for 1 h at room temperature. Blots were then washed with TBST and developed with an ECL detection system (Thermo Fisher Scientific) and exposed to film. Films were scanned, and densitometry analyses of the scanned images were performed using ImageJ. Ponceau-stained total protein was used as the loading control for each blot. For this study, the following primary and secondary antibodies were used: TGF-β receptor 2 [1:1,000 dilution (cat. no. sc-400; Santa Cruz Biotechnology)]; phospho-Smad2 (Ser 465/ 467) (1:1,000 dilution, cat. no. 3101), Smad2 (1:1,000 dilution, cat. no. 5339), phospho-Smad3 (Ser 423/425) (1:500 dilution, cat no. 9520) and Smad3 (1:1,000 dilution, cat. no. 9523), all from Cell Signaling Technology; Smad4 (1:2000 dilution, cat. no. ab40759; Abcam); Smad7 (1:1,000 dilution, cat. no. sc-11392) and Pai-1 (1:500 dilution; cat. no. sc-8979), both from Santa Cruz Biotechnology; and anti-Rabbit secondary (1:10,000 dilution, cat. no. A24537; Life Technologies).

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Smad4 antibody [EP618Y] (ab40759) below.

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