Ion-exchange chromatography - Purification of a transcriptional regulator (Jun/29/2005 )

Hi everybody,

I am a new babe in purifying proteins, please be patient to answer my silly questions. Thanks!

I am going to purify a novel bacterial protein from crude protein extract. The only thing that I know is that the protein is a DNA-binding transcriptional regulator. I have performed ammonium sulfate precipitation, then I want to proceed into cation exchange chromatography.

I am using a 1ml HiTrap SP column (Amersham). I would like to ask...
1) Is the volume of sample being injected a matter? For example, if I have a dilute sample of large volume (10mg/ml, 5ml) compare with a more concentrated sample of small volume (30mg/ml, 0.5ml), which one is preferred? Or in the latter case, will the resolution be greatly decreased?

2) I am now just trying a buffer with pH described by other papers, should I really go and make buffers with different pH? Or if the one that I get can fortunately elute my protein with the salt gradient is already good enough?

3) After fractionation, if time is not sufficient for immediately removal of salt, how should I stored the protein (with high salt in it)? Should I just keep them at 4oC but not -20oC? I afraid at -20oC, the salts will damage my protein.

Thanks a lot!

In my experience (not extensive but reasonable), I have found that the volume isn't so important as
the running speed. Run at a slow rate and you'll get good separation even with large volumes.
Effects of pH will change with proetein but generally the pH of the buffer you load your sample onto the column with is fine. Change in Salt conc. should suffice to give good separation.

In my experience (not extensive but reasonable), I have found that the volume isn't so important as
the running speed. Run at a slow rate and you'll get good separation even with large volumes.
Effects of pH will change with proetein but generally the pH of the buffer you load your sample onto the column with is fine. Change in Salt conc. should suffice to give good separation.
Storing protein is a controversial issue. It is best to dialyze and continue on to next step as soon as possible. But, I have flash frozen my proteins in high salt with liquid N2 and this works well.
BUT, it does seem that I have lost some activity of one of the complexes I have purified along the way. Is this due to the freezing in high salt? I dunno.

Have you dialysed your protein solution after the ammonium sulphate precipitation (I guess you've already have). It is very important to remove
any excessive salt before loading the ion-exchange column.

I guess mikew is right, sample volume is not an important issue in
ion-exchange column. Just make sure that you are not overloading
the binding capacity of the column itself. I would personally go for the
low concentration, bigger volumn sample. The speed of eluting is even
more important than loading, you want to make sure you protein of
interest comes out in a 'narrow' fraction to maintain good resolution.

Oh, yes, by the way, just go for the pH in which your protein is
most stable, unless it is drastic like pH4 or pH9.5. Proteins may stay
in high salt better than low salt in many cases. I sometimes add
10% glycerol and store my protein at -80. It can stay there for
several weeks.

Hi everybody,

I am a new babe in purifying proteins, please be patient to answer my silly questions. Thanks! 

I am going to purify a novel bacterial protein from crude protein extract. The only thing that I know is that the protein is a DNA-binding transcriptional regulator. I have performed ammonium sulfate precipitation, then I want to proceed into cation exchange chromatography.

I am using a 1ml HiTrap SP column (Amersham). I would like to ask...
1) Is the volume of sample being injected a matter? For example, if I have a dilute sample of large volume (10mg/ml, 5ml) compare with a more concentrated sample of small volume (30mg/ml, 0.5ml), which one is preferred? Or in the latter case, will the resolution be greatly decreased?

2) I am now just trying a buffer with pH described by other papers, should I really go and make buffers with different pH? Or if the one that I get can fortunately elute my protein with the salt gradient is already good enough?

3) After fractionation, if time is not sufficient for immediately removal of salt, how should I stored the protein (with high salt in it)? Should I just keep them at 4oC but not -20oC? I afraid at -20oC, the salts will damage my protein.

Thanks a lot!