Gene discovery usually leads to knock down studies etc and one will usually end up requiring to test and optimise the antibody for the protein in question.
Currently, I have western blots to test specificity of the antibody done using cells with Knockdowns.
I am interested to know how the staining will look like in FFPE of these knock downs, so what IHC conditions should i start off with?
Ag retrival (pH 8 or 6?), Ab (1:100 or 1:400)? etc
You should do a low pH first, with say 1:100 antibody, if you get a very week/no staining, then you should go to pH-9 buffer. 15 minutes should do. Normally at this condition, we should get a strong staining; you can then dilute your antibody further if the staining is too strong.
see the paper-http://www.ncbi.nlm.nih.gov/pubmed/24725481
Good point above, also keep in mind some antibody (mabs in particular) work well in some applications but wont in others.
Hi Malcolm,
You may find useful pages 5 and 6 in http://www.ukneqasiccish.org/wp/wp-content/uploads/2014/12/run_106_journal.pdf
or also http://www.ncbi.nlm.nih.gov/pubmed/26280782
Best
Manuel
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