Hi there!
I am studying protein ligand interactions by STD-NMR and the problem is that the imidazole, used in the Ni-NTA puriffication step interferes a lot in my NMR experiment.
I even dialysed it down to nanomolar concentrations, but it was still very appearent in both fluorescence and STD-NMR.
Before going to a pH Ni-NTA ellution method, which could elimate imidazole from my workflow, I decided to try to remove it from my sample using HPLC, but I am afraid that I cannot use acetronitrile because it could denaturate it.
Imidazole's pKa is 6.59. Maybe I could use ion exchange HPLC.
My protein is stable in pH 4-8 range.
I am not sure about what buffers I can use.
Is anyone familiar with cromatography techniques?
It is weird that you still have imidazole after dialysis! Maybe do the dialysis against another buffer!
If you still have imidazole then you can precipitate your protein with ammonium sulfate then re-dissolve it in your buffer. I did it for 2D STD NMR and it was fine.
Have you tried size exclusion chromatography? In addition to purifying according to MW and shape, it is also a more thorough buffer exchange method than dialysis.
I would not recommend using ion exchange chromatography to remove imidazole, because if your protein binds to the column you will need to elute it with a salt concentration gradient and that would bring you back to the situation in which you don't control your buffer composition exactly.
Dear Giuliherme
You can certainly try to perform buffer exchange using desalting which is a subclass of SIZE exclusion chromatography used to ready perform buffer exchange.
You can do it manually (using the PD-10 gravity columns - max volume load: 25.ml) or by AKTA system or similar using a
HiPrep™ 26/10 Desalting GE Healthcare, 17-5087-01
You can find a detailed description of desalting on my blog:
https://proteocool.blogspot.com/p/page3-protein-puriifcation-and.html
ProteoCool n°8 :Buffer Exchange Methods overview and
ProteoCool n°11: Rapid buffer Exchange by PD-10 desalting coloumn
If your protein is stable in the range 4-8, At pH 7 you can certainly use PBS buffer (eg 50mM phospate buffer with 100mM NaCl at pH 7.2 which do not contain any hydrogen atom (which i suspect can interfere with your analysis - I'm right?)
good luck
Manuele
You can try gel filtration "Sephadex S200", anion exchange, or cation exchange after Ni-NTA If imidazole didn't remove by dialysis.
Since your protein is stable at acidic pH, next time you can try eluting it from the Ni-NTA column by lowering the pH of the eluent (to 4.5, let's say), completely omitting imidazole.
Nothing new to add to what has been mentioned here already. With regard to the low pH eelution from IMAC have a read of the original paper maybe (Hochuli et al., 1988 in Nature Biotech). I find it always interesting to read these original papers. If your system is highly sensitive to imidazole I would prefer desalting columns (e.g. 17-1408 from GE for standard 5ml columns) but probably I would go for SEC where you can monitor OD280nm and OD215nm so you can actually see when protein and Imidazole are eluting. Good luck
Peter
First make sure you STD-NMR noisy peak is from imidazole. You don't want to solve an non-existed problem. Do a quick STD-NMR control exp with just imidazole. Then make sure if imidazole somehow specifically binds in your system, not only binds to your protein. Check your buffer recipe. Finally you can think of how to remove the imidazole. Basically, most of the methods are working well mentioned above.
Hey guys! Many thanks for all the tips!
It helped me a lot, I'll just plan another experiment.
Kind regards,
Guilherme
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