2 dimensional western blotting

Hi all

I am currently doing some 2D western. The problem i faced is that the antibody i used is not recognizing the antigens. THe antibody i used is the same as those i used for 1D Western. I am just wondering is there any chemical that is in the 2D buffer which could affect the binding of ab?

Your help is very much needed =)

Thanks very much.

I would suspect more than likely your protein of interest is not entering the 2D gel, before antitobdy not working. Do your western standards stain on the 2D blot?

Rb

YEs. I stained my 2D blot with commassie blue and the proteins were present. I am wondering whether reduce and alkylating my proteins before IEF actually change the conformation of the proteins?

I used 5mM TBP and 10mM for 90minutes and then quench the alkylating with 10mM DTT.