Antibodies from gel slices

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Tue Apr 21 16:35:32 EST 1998


At 13:50 4/21/98 -0700, Dom Spinella wrote:
>OLiver DePayer writes:
>
>> Hi, I have TWO problems that I would be very grateful if people could give
>> me advice on.
>> 
>> Firstly, does anybody have a protocol for raising antibodies using small
>> amounts (micrograms) of antigen purified from an SDS-PAGE gel slice or 

>From Dom Spinella:
>My advice is to immunize rabbits with the directly excised band from
>your SDS-PAGE gel.  Just grind up the gel slice in saline and emulsify
>the whole thing with CFA. The acrylamide itself is a pretty good
>adjuvant. This technique has been around for many years.  

I "second" Dom's suggestion having done it myself.  Like Dom, it was at
least 10 or so years ago when I did it...

In my case, I ran 20 IP reactions of which #1 and #20 used radiolabeled
cellular extracts while #2-19 were unlabeled cellular (same cell type!)
extracts.  I ran the gel as normal, dried the gel *without* fixation and
exposed it to film.  The next day, I lined up the gel with the autorad and
sliced out the the region between but not including the labeled IP surface
protein.  As Dom said, rehdrate in PBS mixed with FA and "flew with it".  I
was plesantly surprised that two weeks I after the second injection (3
weeks later after the first) I had a rather high titer to my antigen as
measured on whole cells.

Good luck,
David

=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



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