Dear Nyet,

The behaviour of His-tagged proteins on Cobalt and Nickel columns is similar (although some will quibble over which is 'better'). 

I can't really answer your question, however, without more information.

Did your protein definitely precipitate, or did your column block? How did you confirm that your protein precipitated? If you loaded a bacterial lysate onto the column did you clarify it first by centrifugation? If not, then the debris may have caused a blockage.

Is your protein aggregation prone? Is it a membrane protein, in which case did you include a suitable detergent in your buffer? 

I'm sorry I cannot be of more help. If there's anything else you can add perhaps we can be of more assistance.

James

Hi, James and Lakshmi. 

All this while I have been using Nickel column to purify my protein. Until recently, I decided to give it a try on Talon column (1mL) as I read that Talon is able to purify protein with better purity than Nickel column. However, no significant peak was observed during the purification. So, as usual, after purification, I washed the column with water and then stored the column in 20% Ethanol. To my dismay, during the storing of 20% Etanol, my column pressure started to shoot high and visible precipitation was observed, causing the pinkish transparent matrix to turn into white pinkish solid.

My protein has not been claimed to be membrane protein. However, I suspect that my protein does share some similar characteristic as membrane protein. Gel like precipitation was observed while I was trying to concentrate my protein. Usually, I will loss a lot of protein during this stage. To obtain more than 10mg/mL of my protein is always a mission impossible for me. At 9mg/mL, my protein seems to have reached a 'saturated' phase and refused to be further concentrated. 

Hi,

Some ideas...

Try to wash your column with detergent and see if protein is eluted. Your protein could be a membrane one or not. It's current to reach a limit of solubility even with soluble one. Try to clean your column with NaOH or follow the rigourous cleaning protocol and see if your protein is eluted. Perhaps you could improve your olubility with low concentration of detergent and avoid precipitation. You concentrate when you performed a nickel/obalt/... column and your protein could just precipitate due to a limited solubility. You can try to load less protein also...

Some information is needed about your protein.  It might not be surprising to see precipitation on the Co IMAC resin if the bound protein was not stripped off but then stored in ethanol which could make the protein even more insoluble especially without any buffer.  I assume you used a slightly alkaline pH for your experiments.  Does your protein have cysteines and is it correctly folded?  Is it possible that your protein, if it has cysteines,  is polymerizing through incorrect disulfide bridging and then creating a gel, especially upon concentration?  If you need reducing conditions then use TCEP for IMAC.  If you want to check for disulfide polymerization then add DTT to a sample of the gel/precipitate with or without 1-2M urea to see if it solubilizes.  It may be possible that impure Co catalyzes protein oxidation  whereas Ni has little effect.  Did you try running the precipitate on SDS-PAGE non-reduced, could be a very revealing pattern?  Do you know if your Ni IMAC purified protein is active?  If your protein is stable to acidic pH's (may need some urea)  then elution at lower pH's, rather than using imidazole, may elute the protein due to protonation of the N in the His ring.