Hi Thibaud,

Imidazole < to 150-200 mM at neutral pH should not interfere in GST purification using classical resin. You may dilute your sample before performing this step.

Good luck

Hi Patrick,

Thank you for your answer, I will diluted my sample before loading on the GST trap.

Have a nice day :)

I agree with Patrick it is often necessary to optimize binding, wash, and elution conditions by varying the concentration of imidazole in these solutions. To find the right concentrations you might try using concentration gradient between 150- 250 or more because increasing the concentration of imidazole in the binding and wash buffers generally decreases nonspecific binding, whereas lower concentrations give stronger affinity interaction.so by gradient elution you can find the right balance.

I follow Patrick Rouimi answer.

Just a quick feedback of my purification, as suggested by Patrick I diluted 2-fold my NiNTA eluated protein before the GST step, everything worked fine. Thank you all! :)

Hi, Thibaud

ischange columns to obtain homogeneGood luck!ous protein.

Some GST isozymes may be inactivated through Ni-chelating column. I have such an experience with GST-Tau19 from Arabidopsis thaliana. Changing resins or metals did not work. So I had to choose GSTrap and conventional ion exchange columns to obtain homogeneous protein.

Good luck!

P.S.

I tried histidine elution instead of imidazole, but it did not help either.