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Hello, I purchased antibodies against Corticotropin releasing factor receptors 1 and 2 (ab59023 and ab75168). I used both antibodies on western blots of cell lysates from Wild type, CRF1 KO, and CRF2 KO bone marrow derived mast cells. There appears to be non-specific binding of both antibodies to both CRF receptor KO cells i.e. a band is present when anti-CRF1 antibody is used with CRF1 KO cells. Can these antibodies bind both receptors? What region of the receptor do they bind? The receptors are fairly similar. Do you have any suggestions for optimizing the blot? I followed the protocols provided by the people who reviewed the products on the website.
Asked on Nov 22 2011
I am sorry that you are having difficulties using some of our antibodies in western blot. From the data on the website, it does appear that in addition to recognizing the bands of interest, for ab59023 a 50kDa band and for ab75168, a band at 48kDa, both antibodies do give non-specific bands at various molecular weights. In your western blots, are you seeing a band at the right size and the non-specific bands or just the non-specific bands? For ab59023, the epitope region is between ******of the human sequence and for ab75168 the epitope sequence is ******of the human sequence. I would be happy to help with some protocol suggestions, could you provide me with the protocol that you using, blocking conditions, antibody concentration and incubations times etc. I will then see if I can make any recommendations that would help. I look forward to your reply and helping you resolve this issue.
Answered on Nov 22 2011