His-tag degraded after purification? - (Jun/16/2005 )
i purified my his tagged protein (~ 55KDa) under denaturing conditions (8M Urea). i ran SDS gel, most of the protein came off at the washes(buffer with 2mM imidazole).only some at elution (buffer with 60mM imidazole).no proteins when i eluted at 200mM imidazole, this confirms tt ALL proteins have been removed off the Ni column. i did a western blot using anti- his tag to detect my protein, and there were no signals for my (purified samples)elution, however there were signals for my crude samples. is it possible tt the his tag became degraded after purification? which results in some of the his tagged proteins not binding to the Ni column hence were washed out in the washes? hmmm..
I have a protein which come out from column only with 2M Imidazole. Try higher imidazole conc. Also I have heared, that in some proteins his tag was hidden by structure, so protein could not keep the column. Anyway, you should test by Western all your possible fraction before and after column , to discower, where is your protein.
i have tried to elute my protein at a higher conc. (200mM imidazole) but there was no abs for the fractions. so i tink all the proteins must've been eluted. i did my exp in denaturing condtions (8M Urea) so i tink the whole protein has been linearised therefore the His tag cant be hidden. hmmm...
i can try to do WB for the fractions but the His-tag seems to be giving me prolems in detection (using anti-
Our lab had a similar problem where the His-tagged protein came out in the first washes. It was as if the protein could not bind to the column. Later we figured out that our lysis buffer contained EDTA and the protease inhibitor cocktail from Roche (complete) also contained EDTA, which chelates nickel (cobalt in our case), preventing the His-tagged protein from binding.
When we repeated the procedure without protease inhibitors and without EDTA, we got the protein to bind and it did not come out in the washes.
You can buy EDTA free protease inhibitors from Sigma or make your own cocktail.