I am a M.Sc. student.
I have successfully expressed my protein in soluble part using a solubilizing tag. I faced problems in Ni-NTA purification, as protein was not binding to the column. So, I purified my protein under denaturing conditions.
My protein does not have any enzymatic activity. How can I confirm that my protein is correctly refolded or not?
Dear Jahnvi Ahlawat
sorry but i miss something:
is it your protein in presence of denaturing agent as 6M urea or guanidine or didi you removed the denaturing agent (performing a refolding)
Because under denaturing condition is quite normal that you protein do not show any activity and is not well folded. The are only few enzymes so stable to maintain their activity also in urea.
i tihnk that if you have problem of purification with a solubilizing tag (MBP?) is because your protein is unfolded and tends to aggregate and so also if the tag prevent the precipitation, do you have high molecular weight soluble aggregates where the tag is not so well exposed.
Did your protein contain some hydrofobic regions? eg transmembrane
? or many cisteines?
best regards
Manuele
There are many ways.. but the easiest way to get fluorescence spectra of folded and refolded protein.. google the red shift and blue shift.. and get your answer with the best of your ease.
For folding confirmation without any activity, X-ray crystallography and CD would be the choice...
If your protein contains tryptophan, monitoring its intrinsic tryptophan fluorescence is one way. Using a fluorimeter, scan the emission spectrum (~300-380 nm) with an excitation wavelength of ~295 nm. Then monitor the emission spectra with increasing amounts of denaturant (e.g. 1, 2, 3, 4, 5, 6, 7, 8 M urea or cyanogen bromide). The maximum emission peak will either shift wavelength or intensity with denaturation. The 50% endpoint for denaturation should be around 3-4 M denaturant if your protein was properly folded.
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