Posted 22 May 2009 - 04:47 AM
Any ideas would be great, thanks!
Posted 24 May 2009 - 02:05 PM
1) If you have too much background, that generally means that you have too much antibody, and are therefore getting nonspecific staining. Try reducing the concentration of antibody and see if that reduces your background.
2) lots of background could be a result of your fixing. If you fix for too long, that could be one possible source of the high background. How long are you fixing for? ... Also, you could try removing the methanol from the fix procedure, as dehydration may (relatively rarely) change the conformation of the protein enough such that the antibody will no longer recognize it.
3) I hate to say anything negative about a company, but Santa Cruz antibodies have a reputation for being less than wonderful. Be sure to preabsorb your antibody (ideally you would use non-transfected cells for preaborption, since these will have all the same proteins except no YFP). If you can borrow another antibody from someone else (or buy one), then try that as well. It's totally possible that the antibody may just be no good. Do you have any confirmation that it even works for IHC?
If all else fails, you could always transfect with a different FP (GFP, etc.) and see if that works.
Let me know if you still have problems.
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Posted 09 June 2009 - 06:50 AM
Thanks very much for that! I havn't heard great things about Santa Cruz either but I couldn't find another YFP ab... also what do you mean by pre-absorb the antibody? I fix for 10 minutes with methanol and then 15 mins with PFA 4%, so I wil try leaving out the methanol this time and use less ab and see how it goes.