amonium precipitation - (Jun/16/2010 )
Which protein needs to be amonium preicipated before loading on the column. I have experience with two proteins and for one I did but for other I did not. As I got the protocol from my colleagues but I did not get the concept....
regards
More detail please - eg. what kind of column?
Ammonium sulphate precipitation is quite good method of protein concentration but within some limits. If your protein of interest is at very low concentration, precipitate would be very delicate and hard to manipulate (you may lose quite a lot of it when removing supernatant etc). Some proteins don't like to be precipitated and may denature. etc.
As for compatibility with chromatography - it's OK to precipitate protein with ammonium sulphate for gel filtration because it would dilute your protein and desalt it anyway, however it's NOT OK to use ammonium sulphate precipitation before ion exchange (unless you dialyse it before loading on the column) because high salt content would screw your separation. I'm pretty much sure that reversed phase column would be OK with ammonium sulphate (it is used for desalting of some compounds) and I think antibody and lectin affinity columns would also be OK with it.
I have to use two columns in conjuction. DEAE column and SP column... After preicipitaion I dialyse the protein with the origianl buffer and will load the protien on the column......
Can you tell me why protein will go to SP column and rest of the stuff in DEAE colum????
best
K.B. on Jun 16 2010, 07:04 AM said:
Ammonium sulphate precipitation is quite good method of protein concentration but within some limits. If your protein of interest is at very low concentration, precipitate would be very delicate and hard to manipulate (you may lose quite a lot of it when removing supernatant etc). Some proteins don't like to be precipitated and may denature. etc.
As for compatibility with chromatography - it's OK to precipitate protein with ammonium sulphate for gel filtration because it would dilute your protein and desalt it anyway, however it's NOT OK to use ammonium sulphate precipitation before ion exchange (unless you dialyse it before loading on the column) because high salt content would screw your separation. I'm pretty much sure that reversed phase column would be OK with ammonium sulphate (it is used for desalting of some compounds) and I think antibody and lectin affinity columns would also be OK with it.
Those are both ion exchange columns - SP is cation exchange and DEAE is anion exchange.
In this case choice of column would depend on both isoelectric point and pH stability of protein of interest ie. you have to work at pH where your protein is stable but has charge so it would stick to chromatography medium (gel).
You can find quite nice introduction to ion exchange chromatography in small e-books you can download for free from GE Healthcare Life Sciences website.