We have major problems detecting sr-b1 in Huh7 cells (liver cells) using the antibody from NB400-104 from Novus Biologicals. Does anybody have experience with this antibody concerning lysis of the cells, usage of PVDF or nitrocellulose membrane? Or maybe someone used a different antibody which works better?
Hi
We have never used this antibody but our own, homemade anti-SR-BI antibody (works on western blots using HepG2 extracts - Arterioscler Thromb Vasc Biol. 2004;24:1-7). I can send you some if you want to try.
Dear Thierry,
thank you very much for your reply and your offer to provide us the antibody. After checking your publication we noticed that the antibody you used is exactly the same that we bought from the company Novus Biologicals, an antibody raised against aminoacids 450 to 509 from mouse srb1. Did you provide the antibody to the company?
Hi
Our antibody has been raised in rabbit against a peptide corresponding to amino acids 496-509 of the murine SR-BI sequence. This antibody has not been provided to a company.
Dear Thierry,
could you please give me details on how you performed the immunoblot, did you block in milk or BSA, diluted the antibody in milk or BSA, blotted onto nitrocellulose or PVDF, how much protein did you load and did you add a reducing agent to the sample buffer and if yes, which, beta-mercaptoethanol or DTT? What were the blotting conditions? We usually do wet blotting 250 mA 90 min or overnight 100 mA.
Thank you so much for your help
Nicole
Dear Nicole
To answer your questions: We use powdered milk (5% for the blocking step and 1% for antibody dilution in PBS/Tween 20 0.05% ). We blot onto nitrocellulose. We try to load 20 to 50 µg of protein under denatured (5' boiling) and reduced conditions (10mM DTT final).
Blotting conditions : wet blotting (regular Tris Glycine buffer with SDS and 20% methanol) 90 volts 1 hour. We have also used the iBlot system from LifeTechnologies and it worked fine.
Hope it helps...
thierry
Thanks a lot Thierry. We will try your protocol and hope for the best.
If you are interested there will be an interesting conference on atheriosclerosis in October in Eilat, Israel mainly organized by our lab (Prof. Bianca Fuhrman, Michael Aviram).
http://www.ias.org.il/
Have a successful week. Nicole
Hello,
I recommend an antibody database available at labome.com. For SR-BI antibodies, you can check this link: https://www.labome.com/review/gene/human/SR-BI-antibody.html. In addition to antibodies from Novus Biologicals, Santa Cruz offers rabbit polyclonal (H-180) (Santa Cruz, sc-67098) used in western blot on human samples (ref: Chen et al, J Investig Med 2014). In addition, Abcam has rabbit polyclonal (Abcam, ab36970) used in western blot on human samples at 1:1000 (ref: Jung et al., Drug Chem Toxicol. 2015). EMD Millipore and BD Biosciences also have SR-BI antibodies for WB. I hope this comment is helpful.
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