Does anyone know how to prepare 4M GuHCl pH 7.4?
I calculated all the desired value it requires 38.2g of GuHCl for 100ml but it's pH is not stable. It starts changing by its own without any addition
If you want to maintain a specific pH, then you should include a buffer. For pH 7.4, you could use Tris or HEPES, for example.
If you want to maintain a specific pH, then you should include a buffer. For pH 7.4, you could use Tris or HEPES, for example.
I agree with Adam B Shapiro . Guanadinium hydrogenchloride on its own has pKa closer to 12, and is therefore going to have no buffering capacity at pH 7.4.
Thanks Adam B Shapiro for your suggestion i tried it but it did not showing good unfolding activity with additional buffer components
If I understand you rightly, Vijay, you are saying that the protein did not unfold when you buffered everything at pH 7.4? i.e. both the protein and the GdHCl were buffered... If that is so, then you didn't get a bad result at all, but rather a very good one. It indicates that any previous unfolding you were seeing was a pH effect rather than a GdHCl effect. This alters how you judge the relative stability of this protein to your standard and other proteins, but at least it is a clear result. The protein is more stable than you think.
Unless you aren't saying the protein didn't unfold or that you had everything buffered. Then it would just be a completely unclear result.
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