Raju,

If you will purify this protein again, you can cut it off from the Co-resin instead of eluting with 250 mM imidazole. Just add TEV (or whichever protease in your vector is responsible for cutting off the tag), incubate with the resin for 30-60 min and wash the protein off the resin with buffer of your choice WITHOUT imidazole.

Vitali

Basically im idazole dos not perturb the native structure of a protein, however you must have to remove imidazole to satisfy the referees and you also... imidazole produce imidazolium in oxidized state which can petrub your system...

to remove imidazole best process is to dialyze the sample.... you can use small volume dyalize tube to avoid protein loss..

Imidazole may or may not disturb your assay. You could try both with and without or you could just do the right thing and get rid of it. You may also have some cobalt leeching off of the TALON which is even more likely to cause problems (if it comes off the column). 

The optimal way of removing it is size exclusion chromatography, but dialysis or centrifugation concentration+dilution are also fine. As are desalting columns. More exotic would be one of those crossflow thingies.

I hope this helps.

I would always try to remove imidazole from your system-it is much easier to do this than to try and exclude any effects on/artifacts in your assay, especially with cells where the effects may not even be obvious until a few days later. Also imidazole can cause your protein to precipitate if it is stored in the IMAC elution buffer for more than a few hours so your effective dose estimates could be way off.

Use a gravity PD-10 column or HiTrap desalt with a syringe  to do this manually and process up to 2-3ml per run, it only takes a few minutes, if your volumes are smaller then there are plenty of spin-column formats that you can use for samples of a few hundred µls.

Losses are generally very low unless you take the salt concentration right down (say below  50-100mM?) then you may be in danger of your protein being retarded/retained on the column. If you have 10s of mls then you could ask around to see if anyone in your institute has something like a HiPrep desalt column that can desalt up to 15mls per run in a few minutes-especially if they have an FPLC available-but even this column can be run manually with a syringe if you have to.

This way you can buffer exchange into anything you like, even your cell media, very quickly and you then also remove any unwanted buffer/dilution effects on your cells.

It could stick to dialysis tubing, or to gel filtration resin in spin columns, you just need to follow separation and do proper controls. Spin columns are very fast and often work very well for buffer exchange but you may need to screen several buffers to maximize recovery. Good luck!

In addition to dialysis and gel filtration or desalting columns, you can use ultrafiltration, either centrifugal units or pressure cells. You concentrate the protein, allowing the imidazole to pass through the membrane, then re-dilute the protein with buffer. Repeat if necessary to further reduce the imidazole concentration.

Raju,

If you will purify this protein again, you can cut it off from the Co-resin instead of eluting with 250 mM imidazole. Just add TEV (or whichever protease in your vector is responsible for cutting off the tag), incubate with the resin for 30-60 min and wash the protein off the resin with buffer of your choice WITHOUT imidazole.

Vitali

These Amicon filters are very nice for buffer swaps and a quick way to remove Imidazole and cobalt that may be in your eluate:

http://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715

I agree with Adam and Terry about the convenience of concentrators but dilute the protein/imidazole mix 5-10X  before first concentration to avoid  possible protein precipitation when concentrated in the presence of high imidazole. Also the imidazole at 250mM can damage the membranes or supports of these concentrators-50mM will be well into the safe zone.

You can do buffer exchange really quick in 5ml desalting columns on AKTA. I think you need to be careful about the purity of your purified fusion protein. Sometimes, one-step Ni or Co column gives you only 90% pure protein and probably a mixture of aggregate/polymer/monomer. I recommend doing an ion exchange column followed by a size exclusion. That will probably give you 99% pure protein in the final buffer you desire if it is compatible with the size exclusion column.

Thank you all for your kind help

Just to update my work,

I have performed using 0.4 gm/ml of Ammonium Sulphate to remove the imidazole from my sample. I used imidazole as control to see if that is precipitating. I hope this would doesn't affect the protein folding

hi researchers,

i have eluted  the recombinant protein with 250mM imidazole. is it mandatory to remove the imidazole before injecting in rabbit? if i inject the protein as such, whether it will influence the antibody production?