How to get clean negative result in immunoprecipitaion? - (Feb/12/2008 )

I did IP with antibody and did western with the same antibody. The other antibody (polyclonal)I have for the same protein from the same species anyway. The antibody used in IP is rabbit antiserum, so I used rabbit serum as negative control.

The protein is 100 kD. There is a strong band of about 100 kD in the IP lane, but the band is even stronger in the serum control. Could 100kD be heavy chain? I think it is unlikely as heavy chain should be 50kD?

To get a clean result, I used Trueblot, which yields no signal either in IP or serum control. So the method did not work for me.

Will you please recommend a rabbit serum that I can try to get a clean result? Or please give me some advice on how to get clean results with negative control?

Thanks a million.

sparrow.

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Not very sure of this point, but anyway, have you precleared your lysate with normal rabbit serum-Protein A beads before you do IP?

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No, I precleared only with the beads. Do you mean conjugate the serum with the beads first, then preclear with the serum-beads complex in both lysate for IP and serum control?

My point is if I IP with one antinody, and then use another antibody from different species in western blotting, I may be able to get clean results.

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