Problems with ChIP

[Denise]

Hi Everyone!!
A fellow researcher has stressed to me that it is very important to empirically determine the proper antibody concentration to use in order to get the best results... so my question is how how would one go about this?

So far I have been adding 5 micrograms and incubating overnite, but I'm not really sure what the negative effects will be if I am adding way too much (maybe i only need 1 microgram?) What would too much antibody do to my results?

Also I do not have a very large supply of my starting material (primary keratinocyte culture), therefore I need to be very conservative in my approach unfortunately, but if i did have an endless supply how would one perform and verify an antibody titer?

Any suggestions... please post!
Thanks

[kiki06]

Hi Denise!
I suggest that if you want to titer the antibody but don't want to use up too much of your chromatin you could always try decreasing the different volumes of: chromatin, dilution buffer, beads, elution buffer, etc. Also you may not be able to run very many PCRs with the resulting material but you don't really need to if you are just looking at one or two genomic regions.

Good Luck