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insoluble protein Low affinity to Ni-NTA and crashes

insoluble protein Low affinity to Ni-NTA and crashes - Protein Science

insoluble protein Low affinity to Ni-NTA and crashes - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 07-16-2009, 05:58 PM
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Default insoluble protein Low affinity to Ni-NTA and crashes



working on a series of insoluble recombinant proteins with a 6xHIS tag (N-term). Solubility analysis has been done and pellet MUST be dissolved in 6M GuHCL.
Following pellet re-suspension for one hour, it is applied to Ni-NTA and subsequently washed in a buffer series containing 20mM Tris-HCl/250mM NaCl/6M GuHCl/1mM Beta-Mercaptoethanol and increasing concentrations of imidazole ranging from 5mM to 500mM.

Protein has found to elute at 20mM Imidizole.
There is the first issue - SEEMS like a low affinity to Ni-NTA.
FYI - The isoelectric point of the protein is 5.7ish and buffers are at 7.5-8.0

Then there is the next and MUCH bigger issue.
Proteins are dialyzed against 20mM Tris, 150mM NaCl, 0.3M Arginie overnight and upon coming in in the morning there is a pretty good amount of precipitate right in the dialysis casette. Spun it down and looked and the supernatant and the precipitate pellet on SDS-PAGE and yep, the protein is in the precipitate.

Now one big thing is that this is a protein fragment so I'm not sure that re-folding is a huge issue.

What I am in the middle of right now is that I loaded the 6M GuHCl dissolved pellet onto Ni-NTA for an hour. Then, ON COLUMN I am running a 6M to 0M UREA gradient (20mM Tris,250 NaCl, 5mM Imidazole + Urea) and THEN doing my washes and imidazole elutions without GuHCl present. Going to run those out on a gel soon and see if it still crashed on column.

So if anyone has any ideas about the low affinity and more importantly if anyone knows of a magic buffer I can dialyze in that would be so appreciated. I have to try and keep it somewhat physiological, detergents are ok.

I know 6M GuHCl is common on Ni-NTA so I don't know.

Thanks
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Old 07-16-2009, 06:52 PM
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Default Re: insoluble protein Low affinity to Ni-NTA and crashes

Sorry, I forgot to mention that I did try dialyzing into a buffer which contained 0.5% Tween as well, so a low concentration did not seem to help. I have seen cases where concentrations as low as 0.01% Tween/TritonX helped but no luck for me.

Tween vs. Triton? I haven't tried TritonX yet but I assumed that if Tween at 0.5% didn't do it Triton might not.
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Old 07-21-2009, 05:55 PM
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Default Re: insoluble protein Low affinity to Ni-NTA and crashes

IMAC buffers generally have 0.5 M NaCl in them. Some proteins are precipitated by this amount of common salt. Not very often, only about 1 in 40 proteins........but could be mistaken for a general insolubility issue if the cells were sonicated directly in IMAC buffer, which is often the case.

Worth a quick look when the above happens - sonicate in water (CFE buffers itself pretty well), then add 1 M NaCl (quarter vol of 5 M NaCl). If your protein then spins out - bingo, just drop the salt and all will be well. In this scenario the subsequent imidazole will NOT give you the same issue as the NaCl did, and thus proceed as normal for the washing and elution.

Good-luck!
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affinity , crashes , insoluble , low , ninta , protein


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