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problem with immunoprecipitation - (Apr/30/2006 )

Would someone please point out what is wrong with my immunoprecipitation experiment?

This is the procedure that I used.
1. make cell lyste
2. incubate cell lysate with primary antibody (mouse) overnight at 4 degree
3. add protein A agarose beads and incubate for 3 hours at 4 degree
4. spin 30sec at 4 degree
5. change pellet twice with cell lysis buffer
6. resuspend pellet in 50ul of 0.1M Glycine pH2.5 and incubate for 10 min on ice
7. spin 2 min at 4 degree
8. add 5ul of 1M Tris pH8.0 to neutralize the pH
9. load the sample with loading buffer onto SDS-PAGE and run
10. transfer to the membrane
11. block with TBST+ skim milk
12. put primary antibodies (rat)
13. wash then put secondary antibodies (anti-rat HRP)
14. put ECL and develop the film.

I loaded the immunoprecipitated sample in one lane and the primary antibody(mouse) in another lane of the SDS-PAGE.
There were two bands each lane in the western blot. I think they may be heavy and light chain of the mouse antibody.
I used anti-rat HRP as the secondary antibodies in western blot and this antibody should not crossreacted with mouse antibody!!

Thanks in advance.

-Minnie Mouse-

should not react : not true, it depends on the quantity of Ab !
why don't you try to use protein G-HRP or protein A-HRP (check the binding profiles, I don't know for rat antibodies) to detect the first antibody used in western-blot?
protein A and G recognizes the native Ab and not the denatured one (used for IP) ( at least not too much). Protein A recognizes the Fc domain, but the binding profile is less broad than the one of protein G. However protein G recognizes also the Fab domain, and you might see a little the heavy and maybe the light chains (but less than your band of interest).