Have run into a similar problem.
Cleared the background color by running 2M NaOH together with 0.2M EDTA.
Better replace BMT with TCEP (1 mM).
Also keep in mind that adsorption is pH dependent, that is the higher
the pH, the better is adsorption.
Many proteins adsorb irreversibly above pH 7.0.
Hi Katherine,
I recommend Zn-IDA Sepharose (Chelating Sepharose Fast Flow, GE Healthcare),
which we have been using successfully for more than 20 years, since the early
days of IMAC:
Skerra et al. (1991) The functional expression of antibody Fv fragments in
Escherichia coli: improved vectors a
Hi,
This is a fairly familiar issue. A hydrophobic protein with thiols often
contaminates IMAC columns and this kind of contamination can be difficult
to remove completely. In the past some of the methods that worked included
trypsin or papain treatment, followed by massive amounts of chelating
ag
Katherine,
You are not alone. I have inadvertently destroyed a GE HisTrap column with high
concentrations of proteins that contain many exposed cysteines. In my case the
Co2+ resin turned a very dark purplish-brown and the protein appeared to have
crashed out on the column. I didn't try to stri
