Immunoprecipitation - (Aug/06/2009 )

What antibody did you use for capture? one for ELISA or flow cytometry, immunohistochemistry? you must take something directed against the native form of the protein, and not against a denatured form as it is often the case in Western-blot.

little mouse on Aug 7 2009, 04:55 PM said:

let me tell you the whole story....
what i did is using my antibody, and mixing with the sample, after that, i added Protein A inside the mixture of my sample and antibody...
after doing the elution, i just run my elution fraction in SDS PAGE and Western blot with the antibody in it, therefore, i put my antibody in one of the lane in SDS PAGE and also Western blot as a control. The result showed that there is something that after IP, appeared in the elution fraction but not the antibody lane.
Then my Boss told me to separate the antibody and the additional band showed in Western blot...

I think that the antibody is ok, since it can capture the right thing for me, but just i don't know a way to separate the antibody and my target protein after IP ...

To prevent antibody from being co-eluted with your target protein, this antibody must be covalently coupled to the solid support you are using. As you only have an small amount of target protein in your sample, you will need an efficient solid support to make the capture work. Dynabeads are small and numerous - giving them a much higher 'availability', i.e. binidng kinetics compared to sepharose/agarose products.

You can use Dynabeads Protein A or G for the capture, see www.invitrogen.com/immunprecipitation and crosslink the antibodies to the beads. See method here: www.invitrogen.com/crosslinking

Another alternative is to couple your primary antibody directly to Dynabeads M-280 Tosylactivated. The antibody will be covalently coupled to the surface, so it will not co-elute.

I hope this helps

Kristina