Reducing agent in Ni column - Ni-chelated resin turn black under 2-mercap (Apr/03/2005 )
I recently run Ni column in the presence of 2-mercaptoethanol. I found that the resin turn black during equilibration with buffer containing 2-mercap. Will this affect the binding of His tagged protein to the resin? Will this has side effect on my protein?
Thanks for your reply.
Check the concentration of 2-mercapto, recommended maximum concentration is 20 mM for Ni-NTA. DTT is not recommended. Personnaly, I use TCEP, which is a more stable reducing agent, more effective and doesn't smell at all (compared to the aweful smell of 2-ME or DTT). I have used up to 5mM TCEP on Ni-NTA columns without affecting the protein binding or yield. But to be safe, 1mM TCEP is what I use frequently.
I used only 10mM 2-mercaptoethanol, it still turned black.
Very odd ! When the resin turns brown/black, it's because the nickel is being reduced. Do you reuse your resin by stripping/recharging it with nickel ? If so, I recommend you use a fresh resin. Also, I really recommend TCEP, give it a try. Finally, I suspect the mercapto concentration is higher than you estimate it. I don't know, maybe someone prepared a bad concentration. In that case, discard all your solutions and start with new ones. I don't see other alternatives, the resin is definately being reduced by something...
The color change of your resin is due to reduction of Nickel. Make APS (ammonium persulfate) solution and add this to your brown resin. It'll re-oxidize your resin. Then, use EDTA to elute any remaining metal impurities and wash with 10CV of water. Now, you can re-use your Ni-NTA resin.
Regarding the side effect of your protein, I do not know. try purify your protein and perform an activity test (if you have it).
Like someone's idea, TCEP is good choice as a reducing agent but, that's expensive reagent. You don't wanna use it too much.