I want to study the stability of one dimeric hyperthermophillic protein at different pH. One way of doing so is to subject pH incubated protein sample to thermal scanning and determine Tm. The other way is to carry out guanidine hydrochloride (GdnCl) equilibrium unfolding studies and derive delta G. But the problem is that this protein requires 4M GdnCl for complete folding and I am aware that the addition of such an amount of of GdnCl will change the pH of the solution, thereby defeat my purpose of monitoring the folding behaviour at varying pH. I am employing GdnCl range 0-5Molar, and using single stock of protein and add all components one by one in a reaction tube. Buffer concentration used is 50mM and NaCl is 100mM. Shall is adjust the pH of GdnCl before adding, lets say for a pH 2 denatured sample, I adjust the pH of GdnCl to 2 and then add it to the protein solution.
Hi!
I would try to buffer the GdnCl stock solutions at the pH you need, e.g. 200mM buffer for a 2x stock for each pH and keep the protein in 5-10mM buffer.
Thermal scanning sounds easier though.
Hi! please check this paper of Garcia-Mira and Sanchez-Ruiz
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1301804/
In many papers this issue is not taking into account 'cause practical reasons. Anyway, the reported free energy values for protein (un)folding have this problem because of the variation in the "true" pH....which influences very differently depending on the physicochemical characteristics of each protein. Hope this help you, cheers!!
I would also go for the thermal melt, it's simpler and provides more flexibilty regarding the buffer conditions you want to test with your protein.
You might want to consider nanoDSF for this, it's a native DSF method that just records tryptophan fluorescence and thus does not even require a reporter dye. So it's absolutely native and you could even work in the presence of detergents.
Please click here for some further information:
http://www.nanotemper-technologies.com/technologies/nanodsf/
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