I am currently working on purifying a phosphoprotein of approx. 25 kDa in length and a pI of 6. In order to purify the phosphoprotein using FPLC, we used a Gallium (III) HiTrap column by GE and an Äkta purifier. Our protein is in a 0.5M NaCl, 50mM Tris pH 6 buffer, while elution is done using a 0.5M Sodium phosphate pH 6 buffer.
Our issue is this: After the first elution, we are unable to re-use the column, either because the phosphate sticks to the immobilized Gallium, or because the elution causes all our Gallium to be washed out alongside it. Since Gallium in solution is colorless, it's hard to tell whether or not the Gallium is still present, and because it is a costly metal, stripping and recharging the column after each purification is also not an option for us.
I have looked far and wide to see what I should do to figure out whether the column can be reused, but so far I've had no luck.
Not really. Even those columns that have been resurrected have a 'very' shortened lifetime (injections) and poor resolution between peaks.
Dear Mr. Neagle,
Thank you for your answer - would you mind clarifying what you mean by " those columns that have been resurrected "? Is this statement limited to Gallium IMAC columns or IMAC columns in general? Because I have been able to perform His-traps with Nickel columns pretty easily even up to 20 times using the same column. If this is the case for you as well, is this only an issue with Gallium IMAC columns or could it be the buffer that is the culprit here? It could be that all the Gallium is still attached to my column, but the phosphates from the Sodium phosphate buffer are strongly attached to the immobilized Gallium and the Gallium thus becomes unavailable for affinity chromatography.
My experience has been mostly C18 reverse phase columns. While they are very stable and have a resilient backbone they can be irreversibly fouled. In your case of immobilization, have you tried an SPE column? They are similar to regular HPLC columns and are used to clean up samples prior to injection (like dye removal). See the Waters, Agilent, and Phenomenex websites.
elution conditions may strip galium (III). in my experience IMAC columns need recharging. it might also be possible to use some other cheaper metal ion
Thank you for your response.
My issue is that the only other metal that has any phosphoprotein specificity at all is Iron (III), as per Machida et al (2007) - but the specificity is much greater with Gallium.
Is there some other elution buffer that doesn't strip the column as much, or will I just have to recharge the column each time I want to purify my phosphoproteins?
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