His-tagged protein co-eluted with associated protein - (Jun/13/2005 )
A colleague is trying to purify a His-tagged protein which has an associated protein that remains bound to it in the Ni-NTA column. What should help to disrupt this protein-protein interaction before eluting the His-tagged protein?
Thanx
Vicky
Maybe you can try increasing/decreasing salt concentration during washing of the column or addition of small amounts of non-denaturing reagents (like the reduced form of Triton, wich does not absorb at 280 nm).
Once people purified protein , and it was copurified with Gro EL protein. Problem was solwed by addition of 3 mM ATP before elution from affinity resin.
Also I have heared that change of E.coli strain for the other, with other protein profile could aid
Thank you for your answers!
Good luck with your experiments 
Also I have heared that change of E.coli strain for the other, with other protein profile could aid
Hi there,
I'm new in the forum, I have just seen about GRO EL and His-tag purifications.
I have a His-tagged protein (in addition to "Protein C and Flag tags on it). I am expressing it in C41 E.coli strain, and it is expressed in low level (you don't see it on commassie when you compare SDS-lysed cells before/after 400 mM IPTG induction) I have tried BL21 codon +, codon 2+ and codon 3+ cells, and no drastic change was observed.
I am trying to purify it using Ni-NTA beads (QIAGEN). After 1.5 hr incubation, I transfer the beads onto a column, wash with 30 mM Imidazole (also tried to include 30 mM Imidazole during the incubation step, which didn't make much of a difference), and elute the tagged protein using 150 mM Imidazole (later, EDTA wash didn't elute a significant amount, so I assume by 150 mM Imidazole, I am recovering most of the tagged protein from the beads)
However, when I run it on gel, and stain with coomassie, I see lots of junk proteins. I have also used different columns to further purigy the protein. Q-column seems to be the best. However, I can never separate it from a ~60-70kD protein in any of the columns (which later turned out to be GRO EL) (I also still had some other minor impurities)
As I have read the forum, I have seen that addition of ATP manage to separate the proteins. Do you know the machanism behind it? And how did you come up with that idea at the first place? Have you read it somewhere? If yes, can you give me the reference (because, it is highly likely that my boss will ask for it). The reason I am asking is that; since GRO EL seems to migrate with my protein in every column, I am assuming that they are associating with each other, do you think ATP will helpto reverse this association, or ATP will only help if GRO EL is bound to the Ni-NTA separately (not in association with the protein)
Thanks very much
exorbitant