Hi there,

As suggested above, what you call a contaminant might be a shorter form of your POI which still contains the His tag (ie opposite extremity is degraded) and presents very similar properties as the full length protein during IEC step. SEC is not resolutive enough to separate such pair of proteins as only a few kD difference occurs.

Hello, SEC will be not a good strategy to increase the purity of your protein in this case. I suggest to use a ion exchanger at another pH and/or in the same buffer with a lower or higher concentration.

It should be interesting to verify the stability of your His-Tag protein as it can be degraded to give another protein with a lower molecular weight than the native one

Hi there,

As suggested above, what you call a contaminant might be a shorter form of your POI which still contains the His tag (ie opposite extremity is degraded) and presents very similar properties as the full length protein during IEC step. SEC is not resolutive enough to separate such pair of proteins as only a few kD difference occurs.

I agree with Philippe that it may be degradation. Can you mass spec both bands?

Also, I would try cleaving off the His tag and putting it back onto your Ni-NTA column (your de-tagged protein would be in the flow through).

Depending on the protein's properties there are affinity purification options that may work. There are ssDNA dn dsDNA columns for purification of DNA binding proteins (Heparin often also works for such proteins). You could also try an ammonium sulfate precipitation, or esoteric columns such as Cybacron Blue (dye-affinity) or Hydroxyapatite.

Thank you for all suggestions.

So, it's likely a degraded product. Actually, after break cell using sonicator (on ice, PMSF was added before and after sonicate), this crude sample already has that significant band on PAGE. It seems that my protein is easily degraded though it is from thermophilic bacteria.

Brian: I may look into different affinity options because sometimes using His-tag (affinity, pre-packed column) alone i managed to get only that 2 bands.

Philippe: I'll try again using IEX because sometimes i managed to get a single band, however only for about 3 elution tube (1ml/tube) (and the band is not thick).

Arthur: I'll try to check that significant band if it is actually part of my protein by cleaving the tag and re-purify.

And..if my protein is prone to degradation, could i possibly add EDTA after the elution step to avoid further degradation? Please advise.

Picture: The single (and sharp) peak still showed 2 bands on PAGE.

Hi

following options might help you :- 

Try using a weak anion exchange like DEAE. with elation by a gradient of pH at constant ionic strength ( choose salt conc. wisely)

and if the above option dose not work try mono Q anion exchanger with conventional elution by increasing ionic strength. I have used mono Q it has tremendous resolution over other matix material.

regards

How much protein do you require ? Do you require enzyme activity ?  Use gradient SDS -PAGE excise band from gel or blot. What happens on non-denaturing gradient PAGE ?  If you get serious -2D PAGE. and excise.  If a breakdown product- protease inhibitor Cocktail Tablets (EDTA free I think)  - Roche ? BIORAD- RotoFor preparative Isoelectric focussing ? beta mercap  in all chromatographic steps.  Just thinking out loud here....  Have fun !  Alarmed at your last comment about sample too dilute

Thanks for the suggestions.

Rahul: i've used DEAE  column, however strong anion  HP showed better result.

Marc: Yes, i need to do enzymatic assay, characterization and reaction.

If your protein is a glycoprotein it could be different glycoforms? Try enzymatic cleavage of the glycans and then SDS-PAGE. You might end up with one band. If not a glycoprotein... How about capillary zone electrophoresis?