Hello, so i am basically doing a time course and seeing the optimal time to solubilise my protein in DDM + CHS. Not really thinking at the time I solubilised my proteins for x hours then when they were done put them in a freezer until all samples were ready to be examined. Would they continue to solubilise at -20C, thus meaning I should restart the experiment?
Thank you
In general, solutes become less soluble in a particular solvent as the temperature is decreased. because the solubility constant is temperature-dependent. The next time you do the experiment, after solubilization for x hours, examine the samples right away instead of putting them in a freezer to be examined later.
If the samples froze when you put them at -20, then any solubilization effect should have stopped. More importantly, when solutions are frozen slowly, solutes tend to get excluded from the ice crystals, leading to concentration of the solutes. This can cause problems once the sample is thawed, and could invalidate your solubilization experiment results. When freezing protein samples, you should use the flash-freezing technique, such as by immersing the sample in liquid nitrogen or a dry ice-ethanol bath. Flash-freezing prevents the separation of ice and solutes.
Hi Jamie
I see another problem, small amounts of protein precipitates may form after thaw procedurę. If time is not long better keep the sample in 4C with protease inhibitor llike PMSF.
Jarek
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