Hi there,

The limitation of affinity purification is the same whatever the tag and its location in the protein sequence :

will the tag be accessible to ensure specific and reversible interaction with the purification matrix?

The answer to this question can't be anticipated unless you have in hand the structure of the protein.

interesting question, I've done a lot of his trap but never the his Tag in the middle of 2 proteins. I'd say that if the HIS is exposed (I mean not hidden in that 3D structure) it should bind.

Thanks Yair,

I also thought that the hist should be exposed for binding. We can however purify the protein with IgG sepharose, but we don't have that column in our lab. I will just give it a try and see if hist tag purification works otherwise we will have to get IgG Sepharose.

Kind regards,

Charan

Hi there,

The limitation of affinity purification is the same whatever the tag and its location in the protein sequence :

will the tag be accessible to ensure specific and reversible interaction with the purification matrix?

The answer to this question can't be anticipated unless you have in hand the structure of the protein.

Dear Dominique, 

Thanks a lot for your answer. I agree with you that the structure of the protein is necessary to see if the tag is not internalized.

Kind regards,

Charan

It is certainly possible. Wether it will work in any particular case of an internal His tag  is not predictable unless you have a structure that shows whether the tag is accessible or not. The only way to know is to try it and see if it works.That's science!

One option is to purify under denaturing conditions (8M urea or 6 M Guanidine HCl) but the disadvantage is that you have to refold the protein after purification. If it is expressed as inclusions you have to do this anyway. 

Good luck Charandeep!

Dear Deepan, 

Thanks a lot for your reply. You are right that it is a hit and trial scenario; I should try to purify my protein and see if it works. 

Kind regards,

Charan