Problems with purifying a secret His-protein expressed in plant - (Apr/16/2008 )
Hi,
I am working with a secret small protein (10Kd) expressed in plant (Tobacco and Arabidopsis). But I have problems purifying it, what we did is to grind the leaves in liquid N2 and homogenized in Denaturing Buffer with 8M Urea. the Kit used is Qiagen Ni-NTA spin column and resin. We do no have problems purifying it form Ecoli. I think the suggested protocol from handbook is not the best for plant-expressed His-protein. Can you suggest me what to do, any input is appreciated.
dshyang 
Hi,
You could try size purifying it and then running it through the NTA column. I had the same problem with mine but mine was a huge protein, so in the end I ran bucket loads of the protein on a large gel, excised the band and dialysed it overnight. I then ran it on a gel and probed it with anti-His to see if it was the right band, which it was, and then purified it through a column to clean up the prep. To further confirm I had the right band I sent it to be Mass Spec'd as well....a long and laborious method, but it worked in the end for me.
You could also try nickel columns from other companies other than Qiagen.
M
You could try size purifying it and then running it through the NTA column. I had the same problem with mine but mine was a huge protein, so in the end I ran bucket loads of the protein on a large gel, excised the band and dialysed it overnight. I then ran it on a gel and probed it with anti-His to see if it was the right band, which it was, and then purified it through a column to clean up the prep. To further confirm I had the right band I sent it to be Mass Spec'd as well....a long and laborious method, but it worked in the end for me.
You could also try nickel columns from other companies other than Qiagen.
M
Check out Dynabeads TALON from invitrogen.
I believe Qiagen holds the patent on Ni-NTA technology. So other companies are simply a 3rd party lincensee for Qiagen's product. Try cobalt and not nickel.