Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

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 (1:1000) -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 (1:1000) -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

Renal protein was extracted from the medulla and cortex of rat kidneys. The protein concentration was measured by the Bradford method, and the supernatant was denatured at 95 °C for 5 min in Laemmli sample buffer. Samples were subjected to SDS-PAGE gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck), which was incubated in the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150mMNaCl, PH = 8.0, 0.01%Tween 20) for 1 h at room temperature and was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam) or anti-Collagen-I (1:1000, sc-293,182, Santa Cruz) or anti-CTGF (1:1000, sc-373,936, Santa Cruz) anti-nNOS (1:1000, 4236 s, CST) or anti-HIF-1α (1:1000, ab2185, Abcam) or anti-VEGF (1:1000,A0280, Abclonal) overnight at 4 °C. Binding of the primary antibody was detected by an enhanced chemiluminescence method (BeyoECL Star, P0018A, Byotime) using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:1000, Proteintech). The quantification of protein expression was performed using Quantity One Analyzer (Bio-Rad).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-HIF-1 alpha antibody - ChIP Grade (ab2185) below.

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