The protein HSF1 (6X-His tagged at the N-terminus) was overexpressed in BL21(DE3) strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. 300mM imidazole was used to elute the protein from Ni-NTA agarose beads. This excess imidazole was later removed by dialysis of the eluted protein sample against a dialysis buffer having no imidazole. When freshly purified protein sample was used for tryptophan fluorescence study, good results were obtained. But protein sample stored in -80 freezer in absence of glycerol didn't yield expected result for the same study. What percentage of glycerol is ideal for storing proteins to be used for biophysical studies (Fluorescence spectroscopy, CD spectroscopy)? Can glycerol interfere with such studies? Should glycerol be removed by dialysis from the protein sample before doing the experiments?
In my experience, glycerol is not essential for storage of most proteins at -80o. What is important is that the protein be frozen very rapidly to prevent separation of solutes from the ice. This means preparing small aliquots and using liquid nitrogen, or powdered dry ice, or a dry ice-ethanol or dry ice-acetone bath to freeze them.
Having said that, it is useful to include 10% glycerol in the samples for storage because it makes it quicker to thaw them out, and may also act as a stabilizer in some cases. I don't think it causes a problem for fluorescence spectroscopy as long as the batch of glycerol is not itself fluorescent due to contamination.
The increased refractive index and viscosity of the solution containing glycerol should be factored into any calculations that require these numbers, such as for dynamic light scattering or fluorescence polarization measurements of molecular weight.
As per my experience, 10-20% glycerol is sufficient for storing proteins. On the other hand, just aliquot your samples before putting it in -80 degree. Repeatedly freeze and thaws affect a lot on the structural integrity of protein samples.
I agree with comments mentioned above. In my experience, even if it is quickly frozen in liquid nitrogen and stored at -80 °C, this operation will still cause irreversible damage to the activity and structure of the protein, especially some extremely unstable proteins. Thus, I use fresh protein for subsequent analysis experiments as far as possible.
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