I have been purifying a His-tagged protein first using Ni-NTA chromatography and then Gel Filtration. After purifying using Ni-NTA, the protein seems to be precipitated after keeping the protein overnight on ice (or even for few hours). The protein was eluted using a gradient of buffer using 500 mM Imidazole (in a buffer of 20 mM Sodium phosphate, 500 mM NaCl, 10% Glycerol, 1 mM DTT). I tried to remove the precipitation by centrifuging but it seems majority of it seems to be completely precipitated (very less in soluble fraction).
Any ideas? Thank you
DTT reduces the Ni-NTA to Nickel solid making a brown precipitate. Did you observe that? Phosphate salts are have low solubility especially at cold temperatures in the cold room so if the sodium phosphate is precipitating, your protein is dissolved in it so it is precipitating with the sodium phosphate. Tris and Hepes are more soluble buffers and are better options and I think Tris is cheaper.
Your protein is least soluble when the pH of the solution equals the isoelectric point of your protein (pI) because the protein is uncharged at the isoelectric point. But for binding to Ni-NTA, the pH of the buffer has to be pH >= 7.8 because at lower pH, the polystidine tag is protonated and so it can't bind to Ni-NTA.
Plug your protein sequence into ProtParam to estimate the isoelectric point:
https://web.expasy.org/protparam/
Try to avoid having the pH of your buffer being near the isoelectric point.
I agree with Adron, 25mM HEPES pH 7.5-8, 125mM NaCl has always been a good staring point for me in the past. Instead of DTT i would use 0.5 mM TCEP as a reducing agent, which will not affect the nickel.
I would look to purify everything in one go, so immediately after IMAC, do the GF step or screen for a good buffer.
To speed things up i would have a loading buffer with 20 mM Imidazole, a wash step with 50mM and an elution with 250 mM.
That way you can do stepwise loading, washing and eluting using a gravity flow column. Then do a you GF with the eluate.
Adron Ung Thank you Adron for your valuable suggestions. I will change the buffers to HEPES. In literature others have precipitation with my protein of interest but have not proposed any solutions. I will have to optimize the conditions. Have you encountered whether high conc of Imidazole be responsible for the precipitation? Thank you.
Abhishek Bastiray , I have heard some people say that Imidazole affects protein stability. It doesn't affect the stability of the proteins I study, but I am sure it affects other proteins.
I am glad you are using 10% glycerol because it is an antinucleating agent that resists proteins from aggregating together and forming crystals or in most cases aggregating together and forming precipitated protein. People that don't have -80C freezer, store proteins in a buffer containing 50% glycerol in -20C freezers. 50% glycerol doesn't actually freeze so the protein suffers no ice crystal damage. So you can probably even increase the concentration of glycerol to 20%. 30% glycerol might affect the flow rate on your HPLC chromatography system, though.
Most hexahistidine tagged proteins don't bind too strongly to Ni-NTA so 150 mM Imidazole should be enough to take them off when bound to Ni-NTA.
One thing that helps protein stability in general is to include 50 - 100 mM L-Arginine amino acid pH ~ 8 in your buffers. There is some complex physical chemistry going on that supports L-Arginine in buffers resists protein aggregation and keeps proteins folded. I don't really understand it, but it works. That is what I would do.
https://www.sciencedirect.com/science/article/abs/pii/S0378517315002677
One other thing that increases protein stability is to include its natural substrate in your buffers. But that's expensive, and when you include the substrate in your buffers, you can't switch the enzyme on and off. It is always on. Makes functional studies difficult. I wouldn't do that, although it works.
Dear Abhishek Bastiray
why did you add DTT? Did your protein contain many cisteines? Do you know if those cisteines are involved in S-S bond formation?
Do you load the precipitate in SDS-page (reducing vs non reducing) to check if the precipitate is an high molecular weight aggregate formed by aspecific intramolecular S-S bond formation that may happen with the time since the lifetime of DTT is not so long when the sample is exposed to the air?
If you are interested you can find more information about non-reducing SDSpage at the following link:
https://www.blogger.com/video.g?token=AD6v5dyp2LLPNQObE-O7xpUxV02oGcx4RQ0mfxIRjK1x1YHBCYw3Av9JZTXdeO6JXLu6wl6Cd2omOIASqXY7BDpDcvlzrJ8fQSoqXnMyd5Fqcc3vRe-1JCCyISDv7Y-UACmOPKhEV6M
at minute 8'00''
good luck
Manuele
Adron Ung has a good idea with arginine. I came across a combination of 50 mM arginine and 50 mM glutamic acid. Article A Simple Method for Improving Protein Solubility and Long-Te...
But be aware that it interferes with IMAC.
As other have pointed out the DTT is most likely the problem. Either try not to use it, or use a DTT stable His-tag resin instead.
Ni-INDIGO (an alternative to Ni-NTA), tolerates up to 20 mM DTT, so well within your parameters. Maybe have a look at it:
https://cube-biotech.com/indigo-common-problems-of-his-tag-purification-solved
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