How to purify unknown protein? - (Oct/24/2008 )

I did ammonium precipitation, and found that the interested protein was in the 60-80% fraction. After dialysis, I passed the crude protein through Nanosep centrifuge column (10K and 30K MWCO). The result showed that the activity remained in the retentate of 10K MWCO and in the eluate of 30K MWCO.

1. Is this mean that my interested protein has MW between 10 and 30 KDa?
2. If I would like to do gel filtration (size exclusion) which media (Biogel, sepharose, sephasex, or else) should I use?
3. If I would like to do ion exchange purification, how to determine whether anion or cation column should I use?

1. yes, most likely.

2. superdex-200 (maybe 75) or sephacryl s-100 or 200.

3. since you don't know anything about the protein (eg pI) then you should try both. but, try anion exchange first. most proteins have a pI in the range of 4-7, so anion exchange at physiological pH would be the best place to start. if the protein doesn't bind then cation exchange can be tried under the same conditions. there is actually a scheme for determining proper pH, [salt], etc for binding and eluting. you can find it in the ion exchange manual offered by ge healthcare (free for download), attached here.