Anti-Caveolin-1 antibody - Caveolae Marker (ab2910)

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 (1:4000) -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 (1:4000) -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

Protein samples were mixed with NuPAGE LDS Sample Buffer 4 × (Life Technologies) and heated at 80 °C for 10 min. Equal amount of proteins were loaded and separated in a NuPage 4–12% Bis Tris gel (Life Technologies) at non-reducing (in the case of Sulfo-N-hydroxysuccinimide-SS-Biotin-conjugated proteins) or reducing conditions, then transferred onto a polyvinylidene fluoride (PVDF) membrane, followed by blocking in TBS 0.05% Tween 20 containing 3% BSA, and incubation with Streptavidin-Peroxidase polymer (1/10,000) (S2438, Sigma-Aldrich) in TBS 0.05% Tween 20, 3% BSA. Loading controls were obtained by labelling PVDF membranes with 0.1% Coomassie R350 (GE Healthcare) in 60% methanol in water during 1 min, followed by destaining in 10% acetic acid–50% ethanol in water. Caveolin-1, CAIX, HIF-1α, HIF-2α, α-tubulin and actin were probed after membrane blocking in TBS 0.05% Tween 20 containing 5% milk by incubation with the following primary antibodies overnight at 4 °C: Anti-caveolin-1 ab2910 (1/4,000; Abcam), anti-CAIX M75 (1/200; BioScience Slovakia), anti-HIF-1α #628480 (1/1,00; Genetex), anti- HIF-2α ab199 (1/500; Abcam), anti-α-tubulin ab7291 (1/10,000; Abcam), anti-β-actin ab8227 (1/5000; Abcam), and then incubated with horseradish peroxidase conjugated anti-rabbit (1/10,000) (7074, Cell Signalling Technology) or anti-mouse IgG (1/10,000) (19044, Sigma-Aldrich) secondary antibodies. Protein bands were visualized by enhanced chemiluminescence (ECL) western blotting substrate (Pierce), and their intensities were measured by densitometry using ImageJ software.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting Caveolin-1 using Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) from Abcam.

Manufacturer protocol

Download the product protocol from Abcam for Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting Caveolin-1 using Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) from Abcam. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.