ion exchange chromatography - (Jul/17/2008 )
hi all,
i am trying to get rid of some high molecular weight impurities in my protein sample which remain after affinity purification on a his bind resin. i am performing this purification on a large scale and have been looking into using anion exchange chromatography on a AKTA purifier to get my pure protein. i was just wondering what buffer would be suitable for a protein with a pI of 4.95 (could i just use Tris at pH8 and if not why?) on a hitrap Q HP column? any ideas?
thanks in advance for your help!
-proteinZ!-
QUOTE (proteinZ! @ Jul 17 2008, 09:38 AM)
hi all,
i am trying to get rid of some high molecular weight impurities in my protein sample which remain after affinity purification on a his bind resin. i am performing this purification on a large scale and have been looking into using anion exchange chromatography on a AKTA purifier to get my pure protein. i was just wondering what buffer would be suitable for a protein with a pI of 4.95 (could i just use Tris at pH8 and if not why?) on a hitrap Q HP column? any ideas?
thanks in advance for your help!
i am trying to get rid of some high molecular weight impurities in my protein sample which remain after affinity purification on a his bind resin. i am performing this purification on a large scale and have been looking into using anion exchange chromatography on a AKTA purifier to get my pure protein. i was just wondering what buffer would be suitable for a protein with a pI of 4.95 (could i just use Tris at pH8 and if not why?) on a hitrap Q HP column? any ideas?
thanks in advance for your help!
Use salt gradient prepared in tris 8.0 is fine.
-genehunter-1-
If the difference in MWts is large enough, and the volume is small enough, you could centricon using a membrane with a cutoff between the two MWts.
Alternately, concentrate the whole thing and separate on a size exclusion column.
-swanny-