Recombinant Anti-Cytochrome C antibody [EPR1327] (ab133504)

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 (1:3000) -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 (1:3000) -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

A549 cells were transfected as described above. Cells were harvested and lysed for 10 min in ice-cold lysis buffer [50 mM Tris-HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2 and 0.5% Nonidet P-40 with complete protease inhibitor cocktail (Roche, Mannheim, Germany)]. The lysates were further centrifuged at 12,000 × g for 15 min at 4°C and the supernatants were then collected in new tubes. The protein concentration was measured using a bicinchoninic acid protein assay kit (Dingguo, Beijing, China). Protein samples were boiled for 5 min in the presence of 5X SDS-PAGE loading buffer. Equal amounts of proteins were subjected to 12% SDS-PAGE and then electrotransferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h in Tris-buffered saline (25 mM Tris at pH 7.5, 150 mM NaCl and 0.05% Tween-20) containing 5% nonfat milk powder, and incubated overnight at 4°C with the indicated antibodies. After washing, blots were incubated for 1 h at 37°C with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. Following further washing, the blots were revealed using the ECL Plus detection system (Thermo Fisher Scientific, Inc.) under conditions recommended by the manufacturer. Images were captured directly by the Gel 3100 chemiluminescent and fluorescent imaging system (Sage Creation Science Co., Ltd., Beijing, China). Quantification of band densities was performed using Quantity One software version 2.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with normalization to the GAPDH signal. The level of proteins of interest in the siRNA group was expressed relative to that in the control siRNA group.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Cytochrome C antibody [EPR1327] (ab133504) below.

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