In lab. working with protein samples (solution in room temperature) cause some problems so we use ice and put the 1 ml and 2 ml vials on ice, the half or 2/3 of the plastic vial is upper than the surface of ice the so it might take some time to reduce the temperature of sample, measuring the T it would be around 4 °C and not lower and even it is 8 °C. Therefore, on ice incubation has a range of 4 – 8 °C is it correct? How much time is needed to cool the 1 gr sample to 4 °C or dose it gets lower, (usually 5 min on ice is considered to be enough for thermal equilibration of sample prepared in room temp) ?
because more than that would not be suitable for enzyme stability or reproducibility of data.
In my experience, keeping the tubes in ice is sufficient. However, if you want to make sure the tubes are at 0oC, make a slurry of ice and water. The temperature is 0oC, and the more complete contact with the tube surface improves heat transfer. You have to be more careful about getting ice water in the tubes when they are open, so you should use a floater or rack to hold the tubes.
Dear prof. Adam,
Thanks. your comments work because liquid water and ice system is around zero, most people use ice without water because it is easier. In this case it seems that the temperature wouldn't get bellower than 4 and it will also get higher. Therefore, on ice without water Temp. is 4 or higher (when room temp. is around 25).
Some also insist that temp near zero is also not good for most enzymes.
Regards
Careful, Yahya Sefidbakht
To most of us, "dry ice" means solid carbon dioxide, which is -78.5°C, not water ice without added water!!!
Dear Prof. Honegger
Thanks for your comment, yes , the ice without water is correct.
Dear Yahya
If your protein sample is a pure then exposing to 4-8 C for a short time will not affect your sample, if your sample is a crude extract then you must have protease inhibitors added to avoid degradation (keep sample on ice). if you are trying to do an assay on ice then do it in cold room (4 C).
Hope this helps
Dear Prof. Albaser
Thanks for your comment, working with crude samples makes it worse, generally cooling with ice needs isolate container and for short times. Once working with luciferase extract the bioluminescence activity change upon incubation on ice which was due to protein aggregation. especially for large and mesophilic enzymes.
Dear Yahya,
If you can change to glass bottles the temperature in the bottles would be lower as glass leads heat much better.
Dear Yahya,
Ice without water ensures that water never seeps in the tube or makes that completely wet. You can straightforwardly use crushed ice on the work bench. But if you see melting ice, you can drain off water and add some more crushed ice or just replace that. This ensures your enzyme activity. Hope you work in aliquots to prevent frequent free-thaw cycles too.
Good luck
Does this mean that the temperature of ice is actually around 4°C - not 0°C - when exposed to room temperature unless it has melted to a considerable extent?
Dear Michael,
yes, you told that in short and comprehensive manner, it seems that is the case for incubated samples.
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