Immunoaffinity Columns

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Mon Sep 26 08:57:09 EST 1994


Hi Everyone
I am making an immunoaffinity column for the first time using Pharmacia's
CNBr-activated Sepharose and monoclonal antibodies prepared by a research
associate who has now left the lab. The protocol I am following is from 
'Protein Purification Methods - a practical approach'. In this protocol 
and the other literature I have read the recommended amount of antibody 
seems to be between 1-10 mg per ml gel and the optimum volume ratios for
the coupling reaction 1:2 gel:ligand. My question is how crucial is the
concentration of the antibody solution? The protocol I am following 
suggests 5-10mg/ml then using *5* volumes of this to 1 volume gel in 
the coupling reaction. This seems like rather alot of antibody to me
and to be quite honest I don't have that much! If I use 2 volumes of 
antibody solution to 1 of gel with the antibody at 1mg/ml would this 
be O.K. or is it a try it and see kind of procedure with no hard and
fast rules?
I would appreciate any input from people with experience in this area.
Thanks in advance
Emma



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