I am trying to do experiments that require WB using anti human CD44 antibody. The antibodies I have work really well for IF but as far as WB goes I am having a lot of difficulties. I repeat the same conditions and sometimes the antibody works great and some other times it only gives non specific staining. As I always carry a tubulin control I know the amount of protein in each well should be enough. Has anybody had this problem with an antibody before? What could be changing?
Maria,
We used a mouse anti-human CD44 IgG antibody (primary, 156-3C11, Cell Signaling Technology) and HRP conjugated goat anti-mouse IgG secondary antibody (secondary, BioRad, 1:50,000 dilution in 4% milk TBST) to blot CD44 from a human vascular endothelial cell line. It worked pretty well. You can give a try with that primary antibody. Our paper describing this procedure is in press with Pharmaceutical Research (PMID: 23568520). You can check it out for details.
Wish you best of luck with the experiments.
Thank you very much Xuefei! I will look for that antibody and your paper.
Thanks again!
Anti human CD44 [8E2] (Cat # 5640) Cell Signaling Technology works well
See Figure-8b
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
[Note: CD44 has more variants, so its likely you may get additional bands.
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
I too have had difficulties detecting CD44 via western blotting but with canine samples. Goodwin is right, there are many isoforms of CD44, so if you are detecting multiple bands, particularly if you are using a polyclonal antibody, this could be the reason.
Anti-CD44 antibody [EPR1013Y] (ab51037) abcam is very useful for WB, IHC, IFF etc.
Thanks all very much for the suggestions! I will look at all these papers and hopefully I will be able to solve my issue!
Yes Jasmine, I do detect multiple bands, using a policlonal antibody, but what is making me doubt that everything is ok is that sometimes I detect a nice thick band between 90 and 100 Kda which for what I understand is the more commonly present sCD44 (at least in Hela cells) plus the additional bands, but in the same samples and conditions if I run the gel again and do a WB I only detect the additional bands but nothing between 90 and 100Kda...
I have used CD44 antibody for western blot and it really work very well for my experiments . Please check my publication Gupta A. and chellaiah M.A.
I have the same problem, also, in my sample, there was a band between 100 and 200 KD. CD44 is very difficult and complex molecule. there are about 20 isoforms and when I repeat the experiment in the same condition, I get different bands.
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