Salt! Add 0.5 M NaCl! And when you wash and elute, also use 8M Urea/salt buffer with imidazole.

Why are you solving your protein in 8M urea?

Moreover, which kind of column are you using?

I did his-tag purification with this buffer: 8M Urea, 300 mM NaCl, 20 mM Tris pH8, but this should not make a big difference. Are you sure about your resin? I used Ni-NTA-Agarose from Quiagen and this resin is kind of blueish. It happend to me that it was not blue anymore and that means that something happend to the Nickel (sorry don´t know exactly what) and prevents binding of his-tag. A second idea would be to avoid Tris as Tris can remove Ni ions. However, it didn´t disturb my purifications. And a third idea would be to change lysis buffer maybe your inclusion body lysis is incomplete. An alternative would be: 7M Guanidium Hcl, 20 mM Tris pH 7,5, 10 mM DTT this is a more efficient lysis buffer but be careful you cannot load the samples lysed in this buffer directly on a SDS PAGE as SDS will precipitate so you have to dialyse, dilute (at least 1:6) or precipitate the sample. So that´s it, no more ideas, hope I could help you a bit and good luck. Tanja

The protein is being purified in 8M Urea simply because we couldn't make it soluble under any means. We're not using a column we're just binding batch by incubating the Probond resin with the lysate on a rotator.

As for Danny's suggestion the protein is definitely not binding, there is a *very* small amount on the resin, the rest just comes in the flow through. I'm giving it a go with Guanidium HCl now, I'll let you know how it goes!

(Note: The buffers were the basics recommended by Qiagen, who say that 8M Urea is OK! Anyway I'll see how the Gu-HCl goes...)

Well the overnight binding with Gu-HCl didn't make any difference either! :) At the minute this is only from 5ml cultures because we were trying to get it working, so we lysed the pellet from 5ml in 500ul of Buffer A (6M Gu-HCl, 100mM NaH2PO4, 10mM Tris pH 8) by sonication. The lysate was DNase treated and passed through a needle a few times to make it less viscous, then incubated with 50ul of Probond resin (25ul bed volume, which should bind between 25 and 125ug protein by my calculations - should be more than enough for a 5ml culture?). The incubation was o/n at 4 degrees, though I have also done shorter at room temperature. Anyway, the protein is still largely coming out in the flow-through. I've checked the sequencing files and they look to be totally fine, though I'm going to try running out some of the lysate and blotting with an anti-His tag antibody to be sure.

Any other suggestions?

(One thing to note is that the resin is slightly brown instead of blue. I don't quite get this as there are no reducing agents in the binding buffer. I wonder if this is a problem? I've seen people that still get binding under these circumstances though, and as I say there's nothing obvious in the buffer A that should cause this? I know some people say that Tris is bad in buffers for Ni-NTA purifications. Could this be why?)

Cheers

Mike

Sorry, crossed wires I think, the resin is still blue (in the bottle), yet goes brown in the Buffer A/B (Urea or Gu-HCl binding buffers). As I said, not sure why as there is no reducing agent in the buffer *shrug*

Right, my mistake, the resin is not actually brown it was just a trick of the light :) So, now I'm even more stumped as to why the protein will not bind. Am now blotting for the His-tag...

OK so I western blotted for the His-tag and it's fine! So now I'm completely stuck.

Hi Mike

in my experience His-tag at the C-term does not work as well as that at the N-term, I do not know why, I suppose that tag is not completed and 3-4-5 His do not bind to the NiNTA.

I suggest you to scale up your purification (100-200 ml) and performe the purification in batch incubating resin and lysate for 30 min- 1h. After that, run a SDS Page loading: 1) Lysate 2)induced lysate 3) Lysate after resin 4) resin. You should be able to follow the protein induced. Guanidine should work, Urea sometime is too old, use ultrapure UREA 99% . If you start from a larger preparation you should get a bit of your protein. Resin is brown when lysate contains DTT, which protein are you expressing? Can the protein be an oxidant?

The worst is that you are expressing a protein with indestructible structures that cover the tag, I am thinking to proteins like amiloid fibers, prions ....

http://www1.qiagen.com/literature/handbooks/PDF/Protein/Expression/QXP_QIAexpressionist/1024473_QXPHB_0603.pdf

Mike, I note "(well, a very small amount binds, but literally almost zero)"

small sometime is enough, in science we must be content!

you are in the statistics of cases

good work!

Hey Paola

Thanks for the advice. We have tried it with an N-terminal tag as well, this still didn't work very well at all. The resin was not actually brown, that was a mistake of the light :) Anyway, I agree, the only thing I can think of now is to try scaling up the purification and hope that we can get some of the protein. I will let you know how it goes!

Cheers

Mike

I also have this problem --> My his-tag recombinant protein couldn't bind to Ni-NTA. It totally leaked out.

Have you tried "Osmotic shock" before pellet lysis ? (http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html)....

I do hope it might be useful and can solve your problem (BUT it didn't work with my protein)

HI,

even i have a similar problem. i had his tag at C-terminus of my protein, could not get good protein yield after purification.

can you change your strategy , can you generate a protein with N-terminal His tag, if it does not affect your protein activity

Hi everybody

I suggest to perform an hydrophobic/hydrophilic profile of your protein before its expression in E.coli. If your protein has 8-15 hydrophobic amino acids stretch it would be very difficult to express it.

Moreover, ask you if it could forms particles

for technical problems dont forget to read all

http://www1.qiagen.com/literature/handbooks/PDF/Protein/Expression/QXP_QIAexpressionist/1024473_QXPHB_0603.pdf

Have you checked if there are any metal ions (or anything similar) which can form complexes with the his-tags and prevent them from binding at the column. Then a precipitation with 2mM EDTA could help (dialyse with the binding puffer).

Or maybe the his-tag changes the conformation of your protein and by that, the his-tag is not longer exposed but in the "middle" of the protein complex. A simple denaturating precipitation can answer this (well, actually, by your binding puffer seems quite denaturating to me).

All the best,

Szymon

or change the vector...use something which have two different tags...for example use a vector which have both his tag and as well as MBP(maltose binding protein ). one example is psv282. in this way if you have solubility problem then it could also be resolved. here you will have to use MBP column. increase the length of the column too.

Regarding Tris: Tris has primary amine so it acts as a nucleophile therefore it is not advisable to use it in Ni-NTA column as it tris can make a coordinate bond with the Ni2+ ion and then it wont let your protein bind. I would use HEPES buffer. when you go to to second step of purification (ion exchange ) then you can use Tris buffer.

Salt! Add 0.5 M NaCl! And when you wash and elute, also use 8M Urea/salt buffer with imidazole.

Hi Mike,

Was you eventually able to purify the his-tagged protein under denaturing condition? I'm running into the same problem with a his-tagged protein and haven't figured out a solution yet. Any advice would be greatly appreciated! Thank you!

thanks Ranjan for details