Co tends to be more specific than Ni in purification. Alternatively, playing with detergent in resin binding and/or wash buffers, eg addition of 2% Tween 20, can also help to get rid of co-purifying proteins without losing your target protein.

Adding to the previous comments, I have had luck with TALON resin loaded with Cobalt, but I think you probably can strip your existing NTA resins and load Co2+ onto them if you follow the regeneration protocols.

I had similar problems previously. I have tried both Ni and Co but could not capture clean his-tag protein. This clearly evident that when the expression of desired protein is very low. Have tried blotting to see if the protein of interest is still in the pellet at high levels? if so repeat the sonication and load into Ni column. I have done the same with my protein which worked very well.

Good luck.

Cheers

Jagan

If you get a good yield and the only problem is purity, consider adding a second purification step such as anion exchange or size exclusion. The histidine-tag is not a very specific one.

Many thanks for all of your advices. Cobalt or adding a detergent sounds promising. Second purification might be the last (but useful) alternative (chromatography or just elution from SDS-PAGE gel, which I used before).

Apart from purification, detection of HisTag protein in whole cell lysate on western blot membrane is problematic sometimes as well (in a specificity manner). I always used Ni-NTA conjugates. Any alternalives? I am thinking of adding imidazole into washing and incubating buffer for developing...

You could dissolve your cell pellet in 6 M urea for His-tag blotting. It will work. Blotting is for confirming your protein expression only in crude lysate. If you want to test soluble vs non-soluble you need to select different buffers.