Nickel IMAC for His-tag - column goes dark brown! What does this mean?
Posted 10 June 2012 - 01:15 AM
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.
Posted 12 June 2012 - 12:22 AM
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.
Posted 12 June 2012 - 11:51 AM
Posted 12 June 2012 - 01:32 PM
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.
Posted 11 October 2012 - 03:58 PM
Posted 14 October 2012 - 10:52 AM
Posted 31 December 2012 - 07:59 AM