A protein question******

Virginia Dress Dress at biosci.arizona.edu
Fri Apr 22 17:26:45 EST 1994


In article <2p6c9fINNm07 at dns1.NMSU.Edu>, smori at nmsu.edu (Shahram Mori)
wrote:
> 
> 
> Dear netters,
> We have 11 ug of a protein isolated from polyacrylamide gel
> electrophoresis. We are interested in isolating the protein from the gel
> and making antibodies to it. We would very much appreciate if you had any
> input and specially references that would allow us to isolate the protein
> effieciently in order to use for antibody production.
> Thanks a milion for your help.
> cheers.
> Shahram Mori
> Program in Molecular Biology
> Dept. of Chemistry Box 3C
> NMSU   Las Cruces NM
> 88003

Do you want monoclonal or polyclonal antibodies?  For polyclonal we have
done the following:
	1)  Just mash up the gel band as well as you can and inject it into the
rabbit.
					I don't know if this would work for the monoclonal because the animals
do
     react to the acrylamide (works like an adjuvant) and it may be too
much for
     the little mouse to take.  Especially since for monoclonals you inject
in 
     the abdominal cavity.  We just injected the rabbits in the scruff of
the
     neck.

 2)  I've never done this, but I've heard of people doing it.  Transfer
from the
				 gel to a solid support (nylon, nitrocellulose) and implant the whole
piece
     or chop it up and inject it.

 3)  Check the antibody handbook by Harlowe and Lane.

	4)  Electroelute from the gel, you will lose some of your protein.

I'm not sure you have enough protein to make antibodies, check the
recommendations in Harlowe and Lane.  If you know the sequence you could
make
peptides and conjugate them to a carrier.  You'll need to do AT LEAST two
injections to get decent titer of antibody and I always did 3.  For
monoclonals
I think I was injecting at least 100ug at a time, which may have been
overkill.

The Harlowe & Lane book is from Cold Spring Harbor press.  I can get a more

complete reference if you need it.  I think it's even in the Sigma catalog


Good luck,
 
Virginia



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