questions about immunoprecipitation - (Jul/18/2007 )

Dear all
I have 2 questions about immunoprecipitation:
1) the antibody that I have used for immunoprecipitation of a certain protein, can I use it for detection of that protein in the immunoprecipitate by western blotting? or do I have to use another antibody that recognizes a different epitope on that protein?
2) what is the criteria for the control antibody that should be used in immunoprecipitation ? is it a must to use that control antibody or no need to use it at all?
thanks

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kenkyuu, you can use the same antibody but only if it's relatively clean, meaning that you can easily detect your protein without question. ideally, however, a second antibody would be best. for the control antibody most people use an IgG that matches the host that your primary antibody is from. so for example, if you are using goat anti-p53 you use a goat IgG at the same concentration and then pull down with your magnetic/separose protein G. A control antibody is an absolute must. If you don't use it, you would never be able to prove that your immunoprecipitated protein complexes were epitope specific or just non-specific complexes sticking to your sepharose/beads etc.

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If you separate your immunoprecipitated material by SDS-PAGE, then the antibodies that you used to capture your protein will dissociate anyway.

Usually we do our IP, then we run a standard SDS-PAGE and western blot. We use the original antibody (same as that used in the IP) as a primary, and a labelled secondary. If you do a western blot in this way, you might also see the heavy and light chains of the antibodies you used to capture your protien (because they were attached to your protein when you loaded it on the gel, but have no resolved to their expected sizes on the gel). It will be a problem if your protein is the same size (or very close to) the size of the heavy or light chains of the antibodies you used for the IP.

Scott.

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