His Tag protein purification - (Jun/13/2006 )
This is my first post here. I am an Honours student at the Bio21 institute in Melbourne, Australia. I am attempting to purify my recombinant his-tagged protein. I have my column and am activating with nickel chloride. I was wondering if anyone could give me any suggestions as to the most appropriate binding and elution buffers are?
Thanks for your help.
that all depends on the properties of your protein, and what form you would like for the final product. There are some general buffer formulation that can give you a place to start, and many proteins can be purified quite readily. However, sometimes you get a 'nightmare protein' that requires loads of optimization and different buffers and stuff...hopefully you will be lucky
I am sorry that I was unable to get the page to load so I could give you a link...however, if you go to Qiagen's website and get a pdf document for a manual called the 'Qiaexpressionist', you will have some great information and some buffer recipes for various purposes. It's long, but believe me if you use NiNTA columns to purify his-tagged proteins, it has some excellent information.
Thanks for your help. I'm trying to purify recombinant His-tagged human myelin basic proten. Thanks for getting me onto Qiaexpressionist - just started reading through it and seems like it will be very helpful. Fingers crossed that it's not a 'nightmare protein', however, from what I've told and what I've already gone through with the expression it may well be.
I hope it works for you