Trouble seperating tagless protein from Ni-NTA beads

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katiekat
katiekat's picture
Trouble seperating tagless protein from Ni-NTA beads

Hi all,
I am having difficulty isolating a protein from its his tag. We have nailed down the correct vector and can cleave the tag off successfully. However, when we try to purify the cut protein from its tag, we are seeing our cut protein on the nickel agarose beads where the his tag is bound. We have tried introducing some imidazole and tween-20 to the buffer to try to reduce nonspecific interaction, in which case we saw some more of our cut protein in the sup, but at least half is still binding to the beads, which is unacceptable. I also am trying to make sure that any UNCUT protein doesn't separate into the sup as well.

Any suggestions?

Thanks for noticing my post.

AUen
AUen's picture
Lets see if we can figure

Lets see if we can figure this out...
A few clarification questions.
When you say you can see your cleaved protein on the beads what do you mean. Are you adding the NTA-beads to cracking buffer and running the sup out, etc?
If you do a standard elution from the Ni column (ie w/ imidazole) at what concentration of imidazole does the tagged protein eluate?
What enzyme are you using to liberate the tag from the protein, I am assuming TEV?

Lets start from there and get this knocked out.

katiekat
katiekat's picture
Hey, thanks for your reply.

Hey, thanks for your reply.
Yes, I am using TEV to take the His-tag off, turbo TEV to be exact, which allegedly works in "any buffer." The cutting doesn't seem to be the problem though, because it cuts just fine, we can see that in our protein gel.
It takes 400mM Imidazole to elute off our desired His-tagged protein. We tried using a buffer with 20mM Imidazole, 1% Ipegal and 600mM NaCl, typical lysis buffer used in purification. However, then we see both the uncut and the cut protein in our sup, AND both in our beads sample.
What's strange, is now we are even seeing our uncut control completely in the sup and none binding to the beads. This is mystifying because, this is the same buffer we use during the purification to isolate our protein of interest. So now we are thinking of just trying to get back to the basics and make sure that our beads are binding our desired protein under different conditions.
Any other suggestions?
If you are interested in helping further, my email is eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%75%6e%64%65%72%6f%72%69%67%69%6e%61%6c%40%79%61%68%6f%6f%2e%63%6f%6d%22%3e%75%6e%64%65%72%6f%72%69%67%69%6e%61%6c%40%79%61%68%6f%6f%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))
I could certainly use the mindpower!

katiekat
katiekat's picture
Oh, and for your first

Oh, and for your first question, if you are asking if I am equilibrating the beads with my desired buffer, the answer is yes. I do not just pull straight from the ethanol resin. I replace the ethanol buffer with my desired binding buffer.

kriodos
kriodos's picture
I´m not sure what are you

I´m not sure what are you doing. I suppose that you express the protein and lysis the cell. After you add Ni-beds (normal or magnetic) to the lysis sample centrifuge or get it with a magnetic device, and wash it. Them you have the protein binded to the Ni-bed. For release it you cut with TEV turbo protease. The problem is that you don’t release it and your protein reminds binded to beds. Is this ok?. Well you said too taht you are sure that the protein is well cutted becouse you see it in a gel. I imagine that you release the protein with imidazole, and after you did a gel and you find the protein cutted. OK thats can be for different reasons:
1) You don´t add Nacl to the buffer and the protein is unspecifically binded to the matrix. Add them 0.3-1 M salt and it will be out.
2) You detect a cut but is not the His tag. Some times the proteases con not cut the tag because the protein adopt a tertiary structure and it is inaccessible. Then if the buffer its not the adequate the protease can cut in a wrong place. Check the cut by western blot with anti his antibodies.
3) The tev is recombinant. Then when you add to it be inmobilized to the bed until you release your protein and tev with the imidazole. Then if you don’t get hurry and you let it time not frozen the tev cut the protein (its turbo tev) and you see the protein cutted. Try to purify the protein with the beds, after, release with imidazole, dialyze it against the appropriated buffer, cut with tev and clean with the beds for remove the tag and recombinant tev
Good luck