Immunoprecipiation desperate request! - need help with IP (May/03/2004 )

Hi everyone! I have been trying to immunoprecipitate a protein with the only commercially Ab against it. The Ab is not so good not even for WB but still it gives me a good enough signal. I know that an Ab that works for WB does not necessarily work for IP. I have tried changing the detergent I use for IP (currently I use NP40 1%), have decreased NaCl content (currently 150mM)... I am currently using magnetic protein G coated immunobeads from Dynal. I have read that sometimes it could be useful to use a linker antibody between the protein G coated bead and my primary Ab against the desired protein. Is anyone familiar with this method? Can anyone help me? Are there any other possible ways of solving my IP problems? Thanx so much. Emiliano

-mexemi-

QUOTE

I have read that sometimes it could be useful to use a linker antibody between the protein G coated bead and my primary Ab against the desired protein.

Yes it is good idea, due to conformational problems of Ab-Ag association raised from pr-G-Ab interactions, but You must have second Ab vs different epitope or several epitops vs Yours primary Ab. Also why do not try such scheme pr-G-Ag-Ab?
Good luck

-Shubenok-

A spermidine linker might be a good idea

-phdconsult-

I don't think a linker will make much of a difference I'm affraid. When tha antibody doesn't work it doesn't work... Your best bet is to try to increase the input, i.e. using more beads and antibody and lysate and eluting in a smaller volume.

Also, indirect IP tends to give you the best possible binding of Ab to target and is as such especially useful for 'bad' antibodies.Incubate the antibody and lysate first (try 24 hours over night at 4 degrees) then add the beads to capture what ever the antibody has managed to bind to.

Good luck:-)

-Roald-