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E.T 05-13-2010 12:15 PM

non-specific binding - IgG

I am looking at a protein of 50kDa which unfortunately has the same size as IgG heavy chain. So my 2nd antibody recognizes the IgG (in addition the cells were previously treated with a monoclonal antibody which is bound to the cells) instead of my protein of interest.

Do you have any hints or can I simply forget using Western blot for this question?



danfive 05-13-2010 04:25 PM

Re: non-specific binding - IgG
How about a IgG depletion kit?
Or just adding Protein G to lysate, assuming no SDS.

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Bonnie 05-13-2010 04:56 PM

Re: non-specific binding - IgG
I assume the IgG is there because these are IP samples?

If your protein of interest is common enough to have multiple antibodies available, you can use antibodies from different species for the IP (and whatever's already bound to the cells) and for the western. For example, use goat Ab for the IP, then use mouse primary Ab for the western. Then your secondary will be anti-mouse, and it won't react with the goat antibody in the membrane.

I've done this when looking for IP'd proteins where we already happened to have multiple antibodies on hand, and it works great.

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