IP a cross-linked macrocomplex of proteins - Cross-linking and immunoprecipitation (Sep/25/2006 )
Dear all,
I want to perform an immunoprecipitation experiment, using as samples cross-linked whole-cell extracts (with formaldehyde) to study protein-protein interactions. So, my aim is IP a specific macrocomplex (cross-linked) of proteins. However, up to now, according to the results, it seems that Ab is not able to recognize the specific epitope. I guess epitope is hide because of the thick net cross-linker could make. Anybody of you knows something about it?
I wait for your answers.
Thanks in advance
Hi mac 16
try to bind the antibody to a sepharose protein A with Dimethyl pimelidate (DMP).
basically,
incubate the antibody with the solid support over night than wash with PBS and incubate for 45 min with 100 mM HEPES pH 8.5 and 40 mM DMP at RT.
wash and repeat the incubation as above.
wash one time with 200 mM ethanolammine pH 8 and incubate for 2 h RT with ethanolammine 200 mM pH 8.
in the end wash 3
times with PBS.........and the solid support with the antibody is ready to be used.
for any questions do not hesitate to contact me!
cheers
Angelo
Hi. Have you checked before the efficiency of immunoprecipitation of your antibody in non-crosslinked conditions?
Hi, miguelon,
Yes, I have checked the efficiency of immunoprecipitation in "basal" conditions, and Ab bind to the epitope (I get several bands as analyzed by Western Blot), but when I use the crosslinker, I only get two bands, heavy and light Ab chains, so apparently Ab does not bind to any epitope. Maybe the concentration of crosslinker that I use is too much high, and it could mask the epitope. I don´t Know...
Thank you for your attention
try to bind the antibody to a sepharose protein A with Dimethyl pimelidate (DMP).
basically,
incubate the antibody with the solid support over night than wash with PBS and incubate for 45 min with 100 mM HEPES pH 8.5 and 40 mM DMP at RT.
wash and repeat the incubation as above.
wash one time with 200 mM ethanolammine pH 8 and incubate for 2 h RT with ethanolammine 200 mM pH 8.
in the end wash 3
times with PBS.........and the solid support with the antibody is ready to be used.
for any questions do not hesitate to contact me!
cheers
Angelo
Hi, Angelo
Thanks for your protocol, but my problem is different from you talk about. In fact, I do not have any problem to get a strong enough binding between Ab and protein G (even without employing cross-linker). My problem is that my Ab (linked to protein G) is not able to IP a specific complex of crosslinked proteins. I have already checked that Ab is able to precipitate an specific protein when it is no crosslinked.
I hope my explanation make clearer the matter.
Bye