I am dialysing a 40KDa protein in a 3Kda dialysis membrane. The buffer that my protein is in has some glycerol remaining. So I want to make sure the glycerol is not there anymore. I ran some thermostability CD experiments and I do not see much of a change in secondary structure after the increment in temperature. Can it possibly be that there is still some glycerol remaining and that my protein is being stabilized by it?
Glycerol should pass through a 3 kDa MWCO membrane, but it may not do so perfectly. Re-diluting the sample after concentrating it, then concentrating again may improve the glycerol removal.
Secondary structures can be very stable in some cases. You may not have reached a high enough temperature to denature it.
Thank you for answering my question.
I increased the temp up to 90 C. I thought that was high enough. I am afraid if I go high it might evaporate.
You could add a chemical agent , such as urea, or try changing the pH, to reduce the thermal stability.
Yes, I use a 3MWCO dialysis tubing to remove glycerol. It depends on the amount of glycerol you have - for example, one of my elution buffers contains up to 2% glycerol and it's dialysed over several buffer exchanges to remove residual glycerol.
Otherwise, glycerol is good as it stabilises intraprotein interactions.
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