2 replies to this topic

[Sandy30]

Question for all the experts in immunoprecipitation: I am immunoprecipitating cell lysates and then running on a gel, and running a Western blot procedure. Can I use the same antibody to capture my protein and for  the Western primary Ab incubation?
Also, has anyone had any luck immunoprecipitating proteins from mouse serum?
Finally, has anyone immunoprecipitated sclerostin (SOST)?

Thanks!
Sandy

[than4]

If you use the same antibody for your IP as for your western you will also pick up the heavy and light chain IgG proteins.  So you need to check that they aren't a similar size to your protein of interest otherwise they will totally obscure your band.

Another option is to use a protein-A-HRP conjugate as your secondary.  It will bind to IgG in it's native state - so it will bind to the primary antibody you have put on your membrane, but not recognise the leftover antibody from your IP as it should have been denatured by the sample buffer, boiling and SDS-PAGE.

Sorry, I haven't IP'd from mouse serum or for your protein of interest.

[laurequillo]

than4, on May 26 2010, 05:26 AM, said:

If you use the same antibody for your IP as for your western you will also pick up the heavy and light chain IgG proteins.  So you need to check that they aren't a similar size to your protein of interest otherwise they will totally obscure your band.

That´s right, you can use it, but you have to mind the IgG.