Question (12041) | Human Histone H3 (tri methyl K4) peptide (ab1342)

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Question

I am writing to find out how the specificity of the antibodies I purchased were determined. I bought: Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895) Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) Histone H3 (di methyl K4) antibody - ChIP Grade (ab7766) I also purchased these peptides: Histone H3 peptide (1-8) - tri methyl K4 (ab1342) Histone H3 peptide (1-10) - di methyl K4 (ab7768) Histone H3 peptide (1-8) - mono methyl K4 (ab1340) Histone H3 peptide (1-16) - unmodified (ab7228) I would like to know of any manual dot-blotting methods you have, that will work with these. I have tried to use them in a blot to no avail so far. The proteins did not bind to the PVDF membrane I used. I was attempting a procedure from the millipore company instructions. there is no dot-blotting apparatus in my lab, so I have to do it by hand.

Answer

We do not have a recommended protocol for Dot-blotting but can recommend trying a nitrocellulose membrane, a general manual will be able to help you. Specificity of those antibodies was tested by WB of Histone (ab121) (0.5µg per lane) and with blocking peptides (modified and non modified peptides). In each case, blocking was achieved only when the adequate peptide was added.

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