Ni-NTA elute without imidiazole under native condition - (Oct/14/2008 )

Dear All,

Searching the internet and the forum unfortunately did not gave any results, so I hope I get something in this forum.

Has anyone experience in eluting a His-tagged protein from a Ni-NTA column under native conditions without using imidiazole?

Additional I have a heme in the protein so chelating agents such as EDTA or EGTA can´t be used.

I thought of using DTT (no disulfid bridges) which reduces the nickel ions in the resin.

Any or better suggestions?

With kind regards georg.

-georg-

You could try low pH or maybe ZnCl2. I always use EDTA or imadazole, so I'm just guessing. I doubt DTT would work. Why do you want to avoid imidazole?

-rosewater-

QUOTE (rosewater @ Oct 15 2008, 12:38 AM)

You could try low pH or maybe ZnCl2. I always use EDTA or imadazole, so I'm just guessing. I doubt DTT would work. Why do you want to avoid imidazole?

Thanks rosewater, I think I´ll try ZNcl2 after thinking about what problems it could make.

Imidazole sticks to my heme in myoglobin and if I can elute my proteien with something else than imidazole I don´t need so much washing steps afterwards.

-georg-

You may try elute with histidine solution. The histidine will compete with his-tag in your protein.

-xksoyor-

the only way that i can see to elute without imidazole is to either add chelating agent (eg edta) or reduce the pH to below 3.5. both of these methods will strip the nickel from the matrix so you will have to regenerate after every run.

see the manuals from ge healthcare:

-mdfenko-