pb with concentrating a protein for antibody production

Jim Kami jakami at ucdavis.edu
Sat Oct 4 19:24:45 EST 1997


One method that has worked well for me in the past is to run an
SDS-PAGE, stain with Coomassie, cut out the band of interest, dry and
crush the gel and inject the gel sub-Q. The gel matrix acts like an
adjuvent. Alternatively the protein can be blotted to either a DEAE
membrane for later elution or to nitrocellulose and the nitrocellulose
injected. In either case the antibodies will be raised to a denatured
protein. If you want antibodies to a native protein, just run a
non-denatureing gel system.

Jim Kami
Blue Rose Biotech 

C Pical wrote:
> 
> Hi all,
> I am expressing a protein as a GST fusion, cutting my protein with
> thrombin while it is bound to the resin. Of course I get thrombin in my
> sample. I would like to get rid of thrombin and also concentrate my
> purified protein before injecting rabbits. I tried Centricon-50 (my
> protein is about 66 kD, I know it was a bit risky to try Centricon-50)
> but lost a lot of my protein which actually seems to be stuck on the
> membrane. So I would very much appreciate any suggestion.
> 
> Thanks.
> 
> C Pical



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