post

pesusi at sara.cc.utu.fi pesusi at sara.cc.utu.fi
Thu Mar 17 08:07:32 EST 1994


    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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