Nickel IMAC for His-tag - column goes dark brown! What does this mean? - (Jun/10/2012 )

Hello,

My lab adventures continue it seems! My column is going dark brown!

I have a GE 20 mL HR Nickel IMAC column for purifying my His-Tag protein.
I have recharged the column by running Nickel Acetate 0.1 M in MilliQ water through it - this was fine. I then ran MilliQ water
through the column to flush away any excess unbound metal.

Next I put 50 mM Sodium Phosphate buffer pH 7.4, 30% Glycerol, 10 mM Imidazole, 10 mM Beta-mercaptoethanol into the column.
The column goes from nice green/blue to this ugly brown! What is going on?!

I suspect that the mercaptoethanol is somehow reacting with the nickel? Is this possible?

When I mix some nickel acetate with the buffer - this horrible smoky fog brown forms.

The protocol I am following is this one:
The mercaptoethanol is there to elute unwanted e.coli proteins more easily.

1. Baker, R. T.; Catanzariti, A.-M.; Karunasekara, Y.; Soboleva, T. A.; Sharwood, R.; Whitney, S.; Board, P. G. In Methods Enzymol.; Raymond, J. D., Ed.; Academic Press, 2005; Vol. Volume 398, pp. 540. Using Deubiquitinylating enzymes as research tools.

UPDATE Tue 12th June 2012

I have used MilliQ water to pump off this horrendous brown/black stuff - two column volume, and thank heavens it comes off.
Next I pumped 2 column volumes of 0.1 M Nickel Acetate into the column and followed with 2 column volumes of MilliQ water.

Then regenerated with buffer WITHOUT beta-mercaptoethanol.

The column looks fine, will try run a prep through it now.

I have seen this happen before when sample is loaded, I think it has something to do with reducing agent reducing the column media, perhaps your BME dilution is off (straight from bottle, it's 14.3M). Might want to try TCEP as reducing agent if your protein has cysteines and histidines and needs it.

Thank you vsoy.
The paper states that BME is there to assist in removing e-coli proteins. We shall see how the gel works out on this one.

Yes, it's the BME causing the brown color- reducing agents like BME, DTT etc reduce the nickel present on the column. Some companies have nickel resins that they claim to tolerate low levels of reducing agents, I've seen different ranges depending on the resin so you may be able to try out some different ones if you need the reducing agent while purifying.

even if they state that their resin is resistant to BME and DTT, it is not quite like that. I would never add in the first purification step DTT/BME (I used to do so and my columns got brown). I usually just wash them long (200-300 mL) with MilliQ water. I do not recharge them because you have to was them extensively afterwards. But if you need to recharge a column, you wash with NaOH, water and then Ni-sulphate and then water again (check the protocol of the columns from GE Healthcare, they have nice description of all the steps). In my experience both the Ni-sephareose from GE Healthcare and the Ni-NTA from Qiagen get brown from DTT. But Ni-NTA can resist a bit more DTT (1 mM without any problem)

Andreea

I've had this same problem and contacted GE Healthcare's tech support and it has an easy solution. The brown color comes from the reaction of the reducing agent with the free nickel that is bound to the column. Pre-wash the column with high mM Imidazole elution buffer first and then equilibrate with binding buffer. If you do this, the column won't turn brown.