I have my protein in the 1M NaoH solution. Protein has N terminal His Tag
1. I want to confirm, under this condition my protein will be in denature state.
2. If yes, then how do I refold my protein.
a) dialysis with working buffer (20mM PB + 100mM Nacl)
b) binding of protein to Ni-NTA column and the eluting protein in the presence of imidazole followed by dialysis with working buffer.
Yes you basically have that right.
NaOH is a harsh way to do it. There's some chance it could do chemical damage to your protein, if it's exposed for a long time. Probably will be fine. More typically people would use less chemically reactive denaturants like urea or guanidinium.
Either way of refolding is reasonable. The only problem is that it might not work. Not all proteins know how to re-fold properly under these standard conditions. You may need to screen many different conditions (buffer, pH, ionic strenght, detergents, redox buffer...) to achieve efficient re-folding of your particular sequence.
You can take a look at commercial "re-folding condition screening kits" to get an idea for formulating useful starting points.
you are corrected on it. I recommend the dialysis procedure for refolding.
It is unusual to have protein end up in 1M NaOH. It will likely damage the protein through hydrolysis especially in the long-term.
Can you use urea or guanidine hydrochloride for protein denaturation instead? If not then you will have issues refolding the protein with the methods you suggest in (2a) and (2b): dialysis might not work to reduce the pH sufficiently to obtain correct folding and your protein will not bind to the Ni-NTA column in 1M NaOH very efficiently.
To test whether the protein is unfolded you can use calorimetry or CD spectroscopy to monitor thermal denaturation (however the 1M NaOH could prevent getting reliable results from either). Ideally you also want a refolded control protein to compare...
Unfortunately, without knowing what the protein is and why it is in 1M NaOH, it is difficult to be more informative. However the basic advice is:
1. Get the protein into a physiological pH in urea or guanidine
2. Then refold by dilution or dialysis (or on-column). The final refolding buffer depends upon the pI of the protein and whether you need detergents salts and co factors.
Good luck.
Thanks all,
I can get my protein in guhcl or urea too.
But will protein will bind to Ni-NTA in the presence of 6M guhcl or 8M urea
Yes, the protein will bind to NiNTA in upto 8M urea or 7M GuHCl.
We use binding buffer: 20 mM NaPO4 buffer at pH7.4, 0.5M NaCl, 20 mM imidazole and 8M urea. The elution buffer is the same but has imidazole at 250 mM.
Then refolding by dilution (at least 10-fold but you should optimise) in refolding buffer optimised for your protein.
Use 8M Urea then refold it by stepwise dialysis to lower Urea concentrations. Dialyze for at least 4-6 hours under each conditions. This way protein will have a chance to refold to its native conformation.
Do not use GuHCl if you will run sds-page, use urea instead. You can also do the refolding while the protein is attached to the immobilized ion.
Dear All,\
Any suggestions for refolding buffer. I have dissolved my protein in 20mM PB +100mM Nacl, 8M Urea. And will bind to NI-NTA column. PI of my protein is 7.4 , composition of buffer I was thinking are 20mM PB +100mM Nacl, 500mM ArginineCl, 8% Glycerol, pH 7. I know I have to standardize these compositions, any suggestions are welcome
Hi Mangesh,
If you are going to refold by dilution then refolding at pH 7 will most likely result in high levels of precipitation upon dilution of the denaturant because the pI is 7.4. You need to go at least 1 pH point (more if your protein will tolerate that) higher or lower than the pI to obtain uniform charge over the protein so that charge repulsion prevents the proteins from aggregating. I would recommend trying pH 8.5 (tris) and 5.5 (acetate) to start with and see how the protein folds. Do you have a simple assay to monitor the function of your protein? That's the best way to tell if it is folding correctly.
Good luck!
@Deepan..Oky I will Replace PB with tris and keep ph to 8.5. Further, I don't have any functional assay to check correct folding of this protein. As per my literature survey gel filtration, 1D NMR, CD would tell me folding of my protein, you please add further for this point if any other way u may know to check correct folding of protein.
Hi Mangesh,
In the absence of a functional assay then the methods you mention are suitable. In addition, DLS (dynamic light scattering) gives information about the presence of multimers and soluble aggregates and calorimetry or thermal denaturation CD can be informative about the thermal stability and therefore, uniformity of refolding. There's a huge body of literature on the subject of determination of correct refolded protein and hopefully you can find methods suitable for your purpose.
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