Hello everybody. I am doing IMAC for years now but recently I decided to give cobalt a try (used nickel before) since it is said to bind His-tagged proteins with higher specificity. I properly loaded a HiTrap Chelating HP (GE) with 50 mM CoCl2 and did an IMAC (wash buffer: 50 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 1 mM be-Me-EtOH / elution w/ 250 mM imidazole). When I tried to regenerate the column / strip off the ions the column just stays pinkish and does not turn white again. I tried so far:
50 mM EDTA, 100 mM, 200 mM EDTA, 50 mM EGTA
buffered solutions (pH 7.4) using either HEPES or sodium phosphate w/ and w/o salt. Ions stay bound! The GE support has no clue. Can anyone help?
Thanks,
Chris
PS: The problem only occurs if I am using a reducing agent during purification. In this case I used 1 mM be-Me-EtOH. If I don't apply it to the column, I can strip off cobalt easily. Any ideas?
Hello Chris,
That is how we do it:
- Strip the resin of cobalt ions by washing with 10 bed volumes of 0.2 M EDTA (pH 7.0).
- Wash excess EDTA with an additional 10 bed volumes of Milli-Q H2O.
- Charge the resin with 50 mM CoCl2 solution (10 bed volumes).
- Again, wash with 10 bed volumes of Milli-Q H2O to remove excess cobalt metal ions.
- Equilibrate the resin with extraction/wash buffer (10 bed volumes).
Dear Syed, thanks for the answer. As I stated in my question, I tried this already with no success. Do you have the same type of resin (HiTrap Chelating HP) or are you using Talon? Do you use reducing agents for your target protein?
I am using Talon and don't use reducing agents. Stripping is more efficiently done at lower pH. Maybe you strip the resin with 0.2 M EDTA pH 7.0, 6.0, and 5.0.
I tried using 0.2 M EDTA, pH 7.0 and then 0.5M EDTA, pH 7.0, but no stripping of cobalt. Has anybody used different stripping/chelating reagent?
i have the same problem with cobalt columns (Talon) : they stay pinkish after 10CV 0.2M EDTA pH 7. In comparison, the Nickel loaded column are perfectly white. any clues ?
Hello everybody.
I used HiTrap TALON crude column charged with cobalt ions and the pink color is always the same (I mean during the charge and the stripping of cobalt ions).
Someone said me that the column color is pink and it is not modified during the stripping of cobalt ions.
PS.: I did purification runs without and with reducing agent ( be-Me-EtOH 1mM)
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