Dear all, Thanks a lot for sharing, seems that either a HIC column or AS would work, and that's great, I should give both of them a try.
I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Thank you very much again! Bei 2011-04-12 joybeiyang 发件人： mi...@chem.ucla.edu 发送时间： 2011-04-12 18:34:27 收件人： joybeiyang 抄送： CCP4BB@JISCMAIL.AC.UK 主题： Re: [ccp4bb] methods to capture proteins from cell culture medium 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 ----- From: "joybeiyang" <joybeiy...@gmail.com > To: CCP4BB@JISCMAIL.AC.UK 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 -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
