Protocol for protein isolation from culture media - (Nov/03/2006 )

Greetings,

I am looking to get insight into protocol for pt isolation from cell culture media as one would do to recover an exogenously expressed protein that is exocytosed.

I attempted a TCA precipitation and observed a lot of protein (presumably) precipitated (white) but when acetone was dried the pellet was extremely hard and little would go into solution with 2x load buffer. Each isolation was from 2ml media from a 6-well.

Protocol.

added 25% volume TCA, mix, ice 5min, spin down 12k rpm, wash 2x cold acetone, evaporate, resuspend.



Thanks in advance

I would concentrate the culture medium. how pure does it need to be? is this for a western or something?

I would use a filter (like from centricon) with the appropriate MW cutoff

Following amikins, try ammonium sulphate pptn. This will bring down your protein in one of a number of fractions, depending on how much ammonium SO4 you have. Check out the different threads for specific protocols.
The benefit of this over TCA is that your protein should still be native. The advantage over Centricon is that you get a degree of purification with each step, assuming you want to do something with your protein.

hi



plz any one tell me whts the propers steps for isolating protein from cultural media using amonium sulfate.i h'v got one protocol in net bt they do gelfiltration/de-salting columns for removing extra ammonium sulfate. that i cnt manag it . is there any other method any one knows.

as i h've CHO cell line, n protein comes in 5ml media (60mm plates).

thnx