Protein Isolation

Jeffrey Jackson jeffj at u.washington.edu
Tue Mar 4 17:55:51 EST 1997


On Tue, 25 Feb 1997, Ali McBride wrote:

> Fellow Netters,
> 		I have a question on how to isolate proteins using a sepharose A
> column, that is conjugated to a unknown Ab.  This antibody is a
> monoclonal Ab, as well as being classified as an IgG1 subtype.  I want
> to know what you would recommend in terms of protocols, or papers that
> might help me elude the answer to the problem(No Pun Intended).  I have
> used the protocol from Harlow et al., directly.  I tried to elute with a
> low pH glycine as well as MgCl2.  I tried to elute the protein, however,
> all I found on my coomassie gel were bands correlating to heavy chain
> and light chains of the Ab.  
> If you have any suggestions I would greatly appreciate an email.
> ALI MCBRIDE
> 
> 
You're going to have to be more clear than this.  I might be able to help
you if I knew better what you were trying to do.  Given that, I can come
up with three possible interpretations.

1) You have a sepharose column with a monoclonal conjugated to it and
don't know what the monoclonal is.  If this is the case, then don't waste
your time.  Chuck the column and make a new one with a known antibody.  

2) You have a monoclonal stuck to a protein A-sepharose column and can't
get it off.  Try eluting with 0.1M citric acid.  That works pretty well
for me.

3) You can't get the protein to unstick from the antibody.  If that's the
case, then 50mM Diethylamine will elute pretty well anything.


If you have any further questions feel free to email me back.
HTH,
Jeff 




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