post

[Tony Sanchez]

    Hi!
I have a question concerning antibody coupling to protein A Sepharose CL-4B or
CNBr activated Sepharose 4B columns. I am using polyclonal antisera and tried
and tried to couple my IgGs but not succeeded. Do you have good procedures? 
How
much should I use matrix and antisera? What are the binding capacities of
matrixies? Have you any ideas how to purify my antibody? It is a little bit
unspecific and I do not have antigens to use in purification. Except cell
lysate. What are good conditions to incubate my protein antigen (30 kDa, pI
8,9, produced in baculovirussystem) so that I could bind as much as possible
antigen to antibody and then elute it. What about elution conditions? Has
anybody used LIS lithium diiodosalicylic acid. How to handle with it? is it
autoclavable. What kind of buffers can I use with it. What does it do to my
antibody or antigen? Should I dialyze my samples after elution. Or what is good
way to desalt samples (hopefully cheap!)
Thanks beforehand for your suggestions

   Yours Peter-- 


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Have you heard of a kit from Schleicher & Schuell called AFFINICA.  This kit 
makes a protein A agarose affinity column.  In their protocol they state that you 
can crosslink up to 20 mg of antibody per ml of gel but the highest specific activity 
is with 2-5 mg per ml.  This kit also eliminates the need to purify the IgG's.  I have 
used this kit to make affinity columns from both poly and mono clonal antibodies 
with good success.  Once when using a monoclonal, it was neccessary to purify the 
IgG.  This was done by making a simple Protein A/Protein G column.
I would suggest that you get a copy of the book Antibodies:  A Laboratory Manual  
by Ed Harlow and David Lane from CSH Press 1988.  This reference proved very 
helpful in learning the ins and outs of antibodies.

no connection to the company
==============================================================
Tony Sanchez
University of Florida  Dept. Ophthalmology  1600 SW Archer Road
JHMHC #100284  Gainesville, FL 32610
Phone (904)392-2720      Fax (904) 392-7839           tsanchez at eye1.eye.ufl.edu