I would suggest that you first consider whether these samples are properly folded. If you are observing two different behaviours on ion exchange, this is likely to imply different folding. To assay folding, use either a functional assay if you have one; or if not (as is usually the case), expected behaviour on size exclusion chromatography is a good guide. 

If it turns out that your less pure sample is the properly folded one, then you might consider whether you can do additional purification. If your protein is His-tagged, then you can do purification over nickel whilst the protein is unfolded, which is usually very efficient. If you have no tag, you might consider adding one? I have found that this can be really effective (at the risk of blowing my own trumpet, see the attached paper for an example) - a tag like His-GST-TEV site allows for multiple purification steps on either the folded or unfolded protein, and the GST will assist in keeping the protein in solution. It does increase the number of purification steps, but refolding is rarely easy!

Good luck!

I would suggest that you first consider whether these samples are properly folded. If you are observing two different behaviours on ion exchange, this is likely to imply different folding. To assay folding, use either a functional assay if you have one; or if not (as is usually the case), expected behaviour on size exclusion chromatography is a good guide. 

If it turns out that your less pure sample is the properly folded one, then you might consider whether you can do additional purification. If your protein is His-tagged, then you can do purification over nickel whilst the protein is unfolded, which is usually very efficient. If you have no tag, you might consider adding one? I have found that this can be really effective (at the risk of blowing my own trumpet, see the attached paper for an example) - a tag like His-GST-TEV site allows for multiple purification steps on either the folded or unfolded protein, and the GST will assist in keeping the protein in solution. It does increase the number of purification steps, but refolding is rarely easy!

Good luck!

The easiest way is to change pH. i don't know what pH and what resin you used, however if you change the pH of the buffer you could probably purify your protein. 

Hi Nausheen, generally speaking, expressed proteins deposited into inclusion bodies are mostly denatured globs of protein.

You should check if your expressed protein is functional before spending more time and energy to purify it.  

If you are expressing a protein that is conformationally sensitive, then you should harvest the bugs before your protein is deposited into inclusion bodies. 

In addition to the above comments, you should consider whether you are forming the correct disulfide bonds, or preventing the formation of undesired disulfide bonds, during refolding. Preventing disulfide bond formation is simply a matter of having sufficient reducing agent. Forming correct disulfide bonds is trickier and involves getting the right balance between, for example, reduced and oxidized glutathione.

Refolding on column may increase the yield of correctly folded protein. If your protein is His-tagged this can be done on an IMAC (immobilised metal ion affinity chromatography) column.

It is better option to go through the size exclusion chromatography and check how much amount of monomeric protein or functionally active protein was refolded if it is an enzyme.

Thank you everyone for your careful responses. Sorry I did not get back to you with my observations earlier.

Trying long incubation hours after the refolding step worked well. Much protein was collected at low salt concentration, and it is good in quality as well, ie; i have better purity, activity and yield. Perhaps sufficient incubation time after the refolding step can make a difference.