Are you using metal affinity beads? If so, the normal procedure is to elute with imidazole. Is the problem that imidazole did not elute the protein? How high a concentration of imidazole did you use?

If a high concentration of imidazole won't elute the beads, you can use EDTA, which will remove the metal from the beads.

You can also use low ph buffer such as ph 4. But the protein needs to be stable at this low ph.

You can also use low ph buffer such as ph 4. But the protein needs to be stable at this low ph.

You can also use low ph buffer such as ph 4. But the protein needs to be stable at this low ph.

Echoing what Adam said above, you can also try using histidine. I think that generally histidine works better than imidazole if you're struggling with imidazole alone, but it is also considerably more expensive.

Best wishes,

James

Adam B Shapiro Thank you for your answer!

I'm using nickel-chelating resin, and, well, I did not add imidazole (following the lab denaturing condition purification protocol). I will try to add imidazole and see how the elution will goes.

The issue I faced right now, - I checked protein on the gel and it seems pure, but the concentration is soo low, so I have doubts that I eluted it efficiently

It's new kind of work for me and I really appreciate any suggestions, comments or recommendations. Thank you!

Hai Li Thank you for your comment!! I did use elution buffer at pH 4. How to know is protein is stable at this pH? Thanks!!

James Torpey Thanks for your remark!

I'll try imidazole.

Sincerely,

Oleksandra

The small amount of protein recovered may also be due to poor expression of the protein, to the use of an insufficient amount of the resin to bind all of the protein, or to poor binding of the protein to the resin.

We faced the same issue with proteins stacked in the beads even with 500mM imidazole…tried pH elution based on protein’s pI, and though this improved a bit, not all proteins could be eluted. The game-changer was switching to Sodium Phosphate buffer + low pH + batch purification…and working with Ni-resins instead of cobalt... this manual helped a great deal (https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/305/240/p6611bul.pdf)!

All the best!

Tonui Ronald thanks a lot!

As many other have already pointed out. Imidazole, pH changes or Buffer changes can help here.

We usually go with Imidazole 500 mM but this number can be increased.

This is our protocol for agarose resins, maybe it helps you:

https://cube-biotech.com/purification-using-his-affinity-agarose

Currently i have a membrane pritein which needs over 300 mM imidazole to elute even 500 mM. And still sone residual protein stoll binds. Donyou know why? Thankso

Currently i have a membrane pritein which needs over 300 mM imidazole to elute even 500 mM. And still sone residual protein stoll binds. Donyou know why? Thankso

Sebastian Lühn Thank you!

Just in case you are yet to solve this, I would increase the concentration of the inducer, in addition to eluting using the recommended concentration of imidazole. Assuming your protein is expressed under the action of an inducible promoter such as arabinose. I would say induce with a couple of concentrations of arabinose (e.g., 0.02, 0.1, and 0.2%) in a small scale experiment and compare. Then, try to tweak the elution. I use this (300 mM imidazole, 50 mM Sodium-phosphate pH 8.0, 300 mM NaCl, 0.01% Tween-20) as my elution buffer and always concentrate my protein following elution.

Best wishes.