Bei,
I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column.
Good luck,
Mike
----- Original Message -----
Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] methods to capture proteins from cell culture medium
Dear all,
My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following:
1. directly load the medium onto a ion exchange column?
2. Amonium sulfate precipitation?
3. anyother thoughts?
Thank you very much in advance!
Best,
Bei
2011-04-12
joybeiyang
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Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
