Dialysis is one of the choices.

I often use 20 mM Tris-HCl buffer (pH 8.0) as external fluid for dialysis before the enterokinase reaction.

Dear Anu Jaglan

which is your sample volume?

in general if you are in the 10-50ml range you can concentrate the protein by ultrafiltration (using amicon ultra centrifuge devices or similar) up to 2-2,5 ml and then perform buffer exchange using PD-10 desalting coloumn that can be performed by gravity on your bench with out any specific instrumentation.

you can find a video about desalting with PD-10 at the following link

https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c

alternativelly you can use a most standard approach as dialysis but it is much more time consuming

good luck

Manuele

enterokinase still has cleavage activity in 150 mM Imidazole, but for instance, 500 mM Imidazole results in near complete inhibition of tag cleavage. If your imidazole concentration isn't that high, enterokinase should still be active just a bit slower. Wait some more time. So long as you have calcium then enterokinase should work.

I, for instance, use Sumo protease to cleave a sumo tag off my proteins in a buffer of 500 mM Sodium chloride. It results in complete cleavage of the tag in 2 days at 4 Celsius. Pretty excessive salt but it completely works if you wait a bit and you can see the tag was cleaved by looking at the SDS PAGE gel.

Kim, Y. S., Lee, H. J., Park, S. H., Kim, Y. C., & Ahn, J. (2021). Expression and purification of soluble and active human enterokinase light chain in Escherichia coli. Biotechnology Reports, 30, e00626.

Hi, for fast and easy buffer exchange (desalting) I propose to use small prepacked columns that can be used with a syringe, a pump on the bench or a chromatography system. The column are named GoBio Mini Dsalt 1 mL or 5 mL. See the attached instruction for details how to do.