One part of the explanation may be that the His-tag cleavage was incomplete, leaving some of the protein still containing the His-tag. The portion with the uncleaved tag would elute with the elution buffer while the cleaved portion would be in the flow-through.

The proteolytic enzyme used for cleaving the His-tag should itself contain a His-tag, allowing it to be removed easily after the cleavage step by binding it to the column while the cleaved protein passes through without binding. This protease can therefore be expected to elute with the elution buffer.

Dear Imtiaj Uddin

what are the theoretical MW of your protein with and w/o His tag? Are the digested and not digested form distunguable at SDS-page?

There are 2 possible reason why you can found some protein in the elution peak:

1) Partial cleavage

2) To early elution (not enough was to remove from the coloumn all the non binded untagged protein before elution)

From your chromatogram it seems that at fractiob 28, when did you start the gradient the the absorbance still decrease and it is much higher than the baseline. I suggest to you to perfrom a longer wash with binding buffer until that the Absorbance will decrease up to the baseline and afterwards perfrom the elution. I suggest to you to avoid the linear gradient but go directly to maximun imidazole concentration.

best regards

good luck

Manuele

You should wash longer before eluting - the UV is not down to baseline. Also there should be no need to do a gradient elution in this case - the elution is just to remove material you don't want from the column not to further separat things.

thanks all Bo Pontoppidan Manuele Martinelli Adam B Shapiro