Immunoprecipitation and immunobloting with different antibodies
Posted 16 March 2011 - 01:36 PM
The Rabbit Ab is said to be raised against the AA 1-70 of this protein, and the mouse ab is against the FL of the protein.
I thought IP with different antibodies should precipitate the same FL protein. Why IP with Rabbit and IB with Mouse did not work? Also the vice versa.
Posted 20 March 2011 - 04:34 PM
Posted 22 March 2011 - 07:46 AM
It could be that one (or both) of the antibodies is not very efficient at immunoprecipitating you protein of interest, so you can't detect it on a blot. Are you sure that the band(s) you are seeing on your IP/IB with the same antibody are not heavy chain or light chain IgG?
The band is at about a little more than 75kD. Also the WB of this protein is at this position (I run IP and total lysate on the same gel). Could it be IgG heavy + light chain (75+25kD)?
Posted 27 March 2011 - 03:57 PM
Are you sure that your antibodies will detect native protein like you would have in an IP?
Posted 31 March 2011 - 09:56 AM
Posted 31 March 2011 - 04:01 PM
Posted 31 March 2011 - 05:52 PM
Does the polyclonal recognise denatured protein? You will be better off IPing with the polyclonal and detecting with the monoclonal.
I'm not sure. But it might be the problem. I'll try IPing with polyclonal to see. Thanks.