Dear Sabuj

I think that for answer to your question we need some more details?

Did you refer to a tagged protein (eg Histidine, GST) and are you using enterokinase to remove the Tag?

in this case it dependends from the MW of your protein and the kind of enterokinase that you are using:

if you are using an His-tagged enterokinase

https://www.genscript.com/protein/Z03004-Enterokinase_His_Bovine.html

you can perform a 2nd subctractive IMAC STEP

https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz

I'm usually use this approach it to the purification of His-tagged protein containing a TEV (instead enterokinase) digestion site, because, His tagged TEv could be easly produced in house.

you can obtain an expression clone for E.coli from Addgene

Addgene: pRK793

If your enterokinase is not contain an His tag, you can try

https://www.thermofisher.com/order/catalog/product/R18002?SID=srch-hj-%20EK-Away%E2%84%A2%20Resin%20#/R18002?SID=srch-hj-%20EK-Away%E2%84%A2%20Resin%20

(sincerelly i never tested it)

or in case that the MW of your protein is lower then 20Kda or higher than 60Kda and therefore faraway from the enterokinase MW, you can try yo perform a SEC purification step.

good luck

Manuele

Dear Dr. Manuele Martinelli, I think I should elaborate my question. Thank you for your well explained, detailed and quite effective answer. Your answer will help me a lot, I believe.

My enterokinase doesn’t contain His tag. By the way, I've got my answer from your kind feedback.

Take my cordial thanks.

Sabuj Das