During Immune precipitation is that possible to use same antibody race, which are use for western bloting for detection. E.g Suppose my protein X I.P which race is Rabbit and detected this protein X Rabbit as well posible . Looking forward your suggestion.
Suppose you use a rabbit IgG to immunoprecipitate protein X with protein A beads. Then you boil the beads in SDS-PAGE sample buffer to elute the protein and run it on a gel. You will have a mixture of protein X and the heavy and light chains of rabbit IgG. You may have more IgG than protein X on the gel. Now you use the gel to make a Western blot and probe the blot with a rabbit IgG and a goat anti-rabbit IgG secondary antibody. In addition to protein X, you will get bands for the heavy and light chains of the rabbit antibody from the immunoprecipitation. There's a good chance that the antibody bands will interfere with detection of protein X. If they don't, then no problem.
One way around this problem is to immunoprecipitate with antibody that is covalently attached to the beads so that it doesn't come off when you boil the beads in sample buffer.
Thanks Adam for your suggestion, but some time I had unexpectedly found protein x band in the place of another interacted protein y , and I am generally use also rabbit y IgG for western blot. It may cause for non specific interaction.
I agree with Adam but also want to add that; its depend on the size of your Protein X if it is between 35-70 then I will not recommend using Rab a.b. for both Ip and wb but if your protein are bigger or smaller, you can Ip your extract with NRS (normal Rab serum) first then take the supernatant and Ip again with your specific a.b. (to get rid of unspecific binding). Another solution is if you biotinylate the antibody (for wb step) and add streptoavidin for detection.
Good Luck
Thanks Fuad for your kind suggestion , yes my target protein is 70 kda.
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