Afinity purification with MAb - kits, protocols?

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Mon Mar 17 04:59:23 EST 1997


Dave, 
you could first clean uf your AB using either Protein A agarose 
(elute w/ glycine buffer, pH 2) or immobilized antigen (e.g. on any 
amino-reactive matrix, pre-packed NHS-activated sepharose (1ml) from 
pharmacia worked well in my lab - elute w/ similar conditions). 
control elution in both procedures by A280.
(you could cascade both procedures if you like)
Then immobilize your purified AB to the material of your desire 
covalently or onto protein A. 
You might have to play a bit with it, 
it might happen that your AB wont let go your protein once bound to 
it.
Good luck!
Wolfgang

(no affils)


> Hi all,
> 
> I have a mouse monoclonal antibody -IgG1- (in ascities fluid) wich I
> would like to use to affinity purify the antigen from cell lysates
> using a column method (protein A beads?). Does anyone have
> suggestions for commercially available kits or a reference to a
> protocol that I could look at.  I know the antibody can be readily
> purified with the MapsII kit but I need some system to use so I can
> run my tissue extract over it.
> 
> Responses via email preferred.
> 
> Thanks in advance,
> --
> Dave Pearton
> pearton at u.washington.edu
> +++++++++++++++++++++++++++++++++++++++++++++++++++++++++
> Down he sank in a chair-ran his hands through his hair-	+
>      And chanted in mimsiest tones			+
> Words whose utter inanity proved his insanity,		+
>      While he rattled a couple of bones.		+
>        +
> "The Hunting of the Snark" Lewis Carroll		+
> +++++++++++++++++++++++++++++++++++++++++++++++++++++++++
> 
> 
---------!all possible disclaimers apply!-----------
                                     
Wolfgang Schechinger
University of Tuebingen
email: wgschech at med.uni-tuebingen.de

http://www.medizin.uni-tuebingen.de/~wgschech/research.htm

public PGP key is avilable on request	



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