Antibody Purification

Mike Clark mrc7 at cam.ac.uk
Wed Aug 27 06:35:06 EST 1997


In article <32C1F935378 at rna.bio.mq.edu.au>, Chris Weir
<URL:mailto:cweir at RNA.BIO.MQ.EDU.AU> wrote:
> 
> Hi all,
>   We have an IgM antibody which is difficult to purify from ascites! 
> The problem is that it falls out of solution very easily. Have tried 
> peg purification which usually is an effective method (for IgM's) but 
> the final pellet wouldn't go back into solution ( in carbonate 
> buffer). So I had to use a tris buffer to get it into solution.
> I then passed it through a column (equilibrated with Carb-buffer) but 
> lost a lot of antibody in this step! and then conjugated what 
> antibody was in solution. The trouble is that this conjugate is 
> now precipitating out! Any suggestions
> 
> Chris Weir
> Macquarie University
> Sydney Australia
> 
Chris,
I have come across this problem with a few monoclonals in the past and it is
possible that both pH as well as ionic strength are a problem.  I found that
the best way to store them was either to keep them in bicarbonate buffered
saline (0.9% w/v) and then dilute them into other buffers only when needed,
or alternatively to store them in the presence of a high concentration of
albumin (10-50mg/ml) in PBS. Another possibility is to add betaine to your
buffer which I have found improves ion-exchange chromatography of some
monoclonal antibodies,

Mike Clark,                        <URL:http://www.path.cam.ac.uk/~mrc7/>
-- 
 o/ \\    //            ||  ,_ o   M.R. Clark, PhD. Division of Immunology
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