Bei, How do you concentrate your media? We use tangential flow filtration. If you get a good filter (Millipore Spiral wound TFF is one example) it goes pretty quick, in ~2 hours you should be able to process 4L(concentrate+ dilute several time in buffer). We secrete proteins from insect cells, but something in the media will strip the Ni2+ from our IMAC column if we apply the sup directly. I have tried AS precipitation and peg precipitation from culture media. Both worked fine at the small scale, but when scaling up I ran into 2 issues: 1. You have to add huge amounts of AS (like kilograms), which increases your volume a good amount, 2. my 'pellets' would actually float on top of the media after spinning, which was tough to deal with. I have also tried loading the media onto a large Q column, but that didn't work well for me-fractions were too messy.
I think you best option is to get a good TFF setup, do your concentration/buffer exchange, and go right to your IMAC column Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst 2011/4/13 joybeiyang <joybeiy...@gmail.com>: > > 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
