Hello everybody,
I want to purify a 32 kDa protein with a C-terminal His-tag. I use the Histrap Crude FF and I elute with 500 mM Imidazol (no linear gradient) and I get a quite distinct peak with approx. 1400 mAU and using a chromogenic substrate I can see that my protein is eluted in one milliliter. Because there are some other bands in this fraction I perform a gel filtration afterwards. And after elution there are only three very small blurred peaks (max. 60mAU) which are not really seperated from each other. In one of the peaks (13mAU) there is only little activity and in this fraction there are lots of other (bigger) proteins. So first I wonder where the residual mAUs are lost during the gel filtration and second I don´t know how to get rid of the other proteins. For cell lysis I use additional Tween and Triton and Protease Inhibitor AE-BSF.
It would be great if you can help me!
Hi
I had almost same problems on purifying a binary protein complex, of which one has His6-tag and TEV cutting sequence.
It seems likely that your protein aggregated. Did you check the exclusion volume?
To get rid of other proteins; you could higher stepwise ratio of imidazole buffer (for instance in the first step 10% B 90 %A then in the second 100% B and 0% B ) or try gradient elution. Adding a TEV cleavage sequence after His-tag would be also helpful if the contaminant proteins have an affinity to column. In that case you could do another round of purification using Ni-NTA spin columns. If you are lucky you will have your cleaved protein in flow through and others on the column
Best
Dhiraj Srivastava
Thank you for your help! So I ran SDS-Page with the his-trap flow through, the fractions of the his trap where I found activity and the fractions of the gel filtration peaks. In the flow through the protein was absent, in the single fractions of the histrap I could see in one fraction a protein of the size I was searching for but with various bigger and smaller proteins and in the gel filtration fractions I saw lots of big protein bands and small ones. So usually the gel filtration should separate the big and small proteins so why is this not the case? Is my protein bound to another big protein and in the denaturated SDS-Page they are separated? Or is the separation by size insufficient with my gelfiltration column? Erman Kocak First of all thank you! I also think that a cleavage sequence would eliminate the problem of contaminating proteins but no commercially available C-terminal His-tag expression vector contain such a cleavage site. This is only the case for N-terminal His-tags, as far as I know. But what do you mean with the exclusion volume? The gradient or stepwise elution seems to be another good idea I will try!- Badges
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