Which antibody to use for ChiP? - Monoclonal or polyclonal? (Jul/21/2005 )

Hi all...

I have to make antibodies against a new protein (600 aa Txn factor) to use in tissue ChIP. Are monoclonal antibodies preferred over polyclonal for this purpose?

How difficult is it to make mono or polyclonal antibodies on your own? How long does it take? Is it worth the effort...except saving money?

Is it okay if my protein is GST-fused or should I raise antibodies against the protein alone? Would it be different for mono or polyclonal antibodies?

Thanks in anticipation...

-learn2GET-

I am not sure about how to make the antibodies, but as far as I know, polyclonal antibodies are preferred for ChIP because fixation may mask certain motifs (due to protein-DNA or protein-protein complexes) so having a polyclonal antibody increases the probability that the chromatin-bound protein can be immunoprecipitated. Hope this helps!

-beccaf22-

QUOTE (beccaf22 @ Jul 22 2005, 12:40 PM)

Hi beccaf22,

Thanks for the advice.....do you think it would be okay to raise antibodies against a GST-fusion protein for this purpose (Tissue ChIP)?

-learn2GET-

I think that would be okay, you may have to remove the antibodies that react with GST by using a GST column etc. to purify the serum, but I really don't know much about making antibodies.

-beccaf22-

QUOTE (beccaf22 @ Jul 25 2005, 12:03 PM)

I think that would be okay, you may have to remove the antibodies that react with GST by using a GST column etc. to purify the serum, but I really don't know much about making antibodies.

Thanks....

-learn2GET-

I know Upstate sells both poly- and monoantibody for ChIP assay. I have used both in my ChIP assays.

-pcrman-