Ni-TED vs Ni-NTA - His-Tag purification (Jun/27/2008 )
I have a low expression level protein with a his tag approx 1mg soluble in 1liter of E.Coli culture (10-20mg inclusion bodies), I purify the protein from the medium and get 3 contaminants one at 70kd, one at 25kd and one at 12kd.. my protein is 28kd. I have a hard time getting rid of these contaminants, Ive washed with 25-60mM Imidazole, changed pH from 7,4-5,5, tried Tween, Triton, tris and Phosphate buffers but cant seem to get rid of them.
So I have tried the Protino Ni-TED from macherey nagel which claim that you get much purer protein in comparison with ni-Nta. The Ni-TED is linked on silica matrix and has a clear white color compared to the Ni-NTA agarose which is light blue.
I did one test.. where i made a denaturating prepp in 8M Urea.
I followed the Ni-TED Manual in every point and Purified the prepp with both Ni-NTA and Ni-TED with same set of buffers and what I got is this.
Nothing bound to Ni-TED and I had no protein at all in my elution fractions.
I had lots of protein in my Ni-NTA purified fractions which was approx 50% pure.
So is this a joke or a hoax.. the Ni-TED is useless, It seems it dont bind the his-tags... Im confused. Has anyone else ever tried the Ni-TED!? Does it work for you guys!?
are you sure the ni-ted was charged with ni? it may have needed to be regenerated.
I followed the manual and it said that the resin is precharged with Ni, I also called the company and they said that the color is supposed to be white compared to NTA which is light blue...
I also did a regeneration just in case.. it didnt help at all.
My short experience with protein purification thought me that not all proteins can be totally purified.
I also tested several systems as cobalt and talon, but in the end they all the same.
if u want to obtain only the above protein, u can load it in one big well onto acrylamid gel
then separate it from the other ones. stain it with commasie and then cut it out of the gel and elute it
to a buffer.
1. With the Ni-NTA, use a relatively high amount of imidazole, like 40 mM, in your protein sample when loading onto the resin.
2. Since your protein is not binding Ni-TED, which is supposedly more selective, I am guessing your His-tag is not fully exposed. Your protein may be aggregated/unfolded and stuck to chaperones, which may be those contaminant proteins you can't seem to purify away. You can check this by running a size exclusion column.