Hello everyone,
Recently, I have been experiencing that my proteins (actually, all of the proteins in my group) elute at ridiculously low concentrations of Imidazole. I'm talking about concentrations around 40 mM or even the 20 mM, which are in the binding buffer to begin with.
The proteins usually come from a culture that was supplemented with metals. The binding buffer consists of either sodium phosphate or Tris, 50 mM and pH 7.5 to 8. Imidazole contents here range between 0 and 20 mM. The columns we use are GE HisTrap, Protino Ni-NTA, HisTrap excel and GoBio mini Ni-NTA.
In any case, it's the same picture. The same constructs for protein production had already been used and purified before in a different lab without any of this issue.
At this point I can't really find a solution anymore. I checked the pH of my buffers right before usage and they were fine. Some of the columns have also been regenerated (stripped of the metal ions and repacked) without any improvement.
If anyone had an idea about what else could be possible reasons, I would be genuinely thankful and deeply grateful!
Two reasons why the proteins may not bind to the IMAC columns:
1. The proteins lack the His tag due to proteolysis or premature termination (for C-terminal His-tags).
2. The proteins are highly aggregated. This can prevent binding if the His tags are not exposed on the surface of the aggregate.
I will add to the excellent suggestions above:
- Perhaps your plates are old and the antibiotics have degraded? If you are using bacteria, a strain without the plasmid may have taken over your plates, assuming transient expression.
- Have you tried washing the column with detergents / NaOH / NaCl according to the manufacturer's specifications? the column may be clogged with junk matter, even after the repacking.
- If you are using induced expression, perhaps the inducer has expired? You could try using materials from the lab that gave you the constructs. Also, lowering the inducer amount can improve folding and reduce inclusion body formation.
Good luck!
Dear Karina Broger
i have some questions:
which expression host do you use? e.coli? mammalian cells?
which metal did you supplemented? Nickel? zinc? copper?
and Why? is your protein a metal binding protein?
did you try to perform the same step with-out metal supplementation?
probably i'm wrong, but in case the metal that you supplement is able to bind the histridine tag, the tag is already saturated and it may explain why do not bind the IMAC
best
Manuele
Thanks for all the input.
According to SDS-PAGE, my protein is in fact over-expressed significantly and as they are metalloproteins, one can see the vibrant colours. The column also does change its colour, too, so it apparently does bind, as the flowthrough is merely a light yellow while the column becomes e.g. cherry red.
For each of the column types, they had been completely new and unused. When we regenerate them, we also do CIP with NaOH, EDTA and NaCl.
I also clarify my lysates by filtration through a 0.4 μm filter after ultracentrifugation at 50000 g for 1 h.
The constructs are from our lab, which moved to a different city. We use the same reagents from the same suppliers. The protein that I recover is fairly pure and fully functional as well.
But the whole issue with the metal ions in the medium however, seems plausible, unfortunately...
Thank you all very much!
Is the pH meter used for buffer preparation OK? Could you recheck buffer pH with a pH meter from a different group?
Is the elution fraction with 20 - 40 mM Imidazole relatively pure? If so then there should not be a problem just elute your protein with 40 mM Imidazole. Maybe other groups studying the same protein are using a different brand of Ni-NTA resin that they bought 6 months ago, whereas yours is five years old. Stripping the Nickel with EDTA and recharging the resin with nickel surely cleans your resin, but prolonged stripping with EDTA causes the Ni-NTA to not work as well as it used to when you bought the resin.
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