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 <[log in to unmask]>:
>
> 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： [log in to unmask]
> 发送时间： 2011-04-12  18:34:27
> 收件人： joybeiyang
> 抄送： [log in to unmask]
> 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"  <[log in to unmask] >
> To: [log in to unmask]
> 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
>
> [log in to unmask]