I have stored my leave sample at -80 and want to prepare a soluable fraction for protein purification. I want to ask is grinding it in liquid nitrogen and further dissolving it in chilled buffer does any harm to activity of my protein, due to temperature fluctuation. OR shall i use a electric grinder for grinding my leave samples stored at -80 and omit the step of liquid nitrogen grinding
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The use of liquid nitrogen for preparing the leaves is the best option you can think of and try. Two advantages you will get; the sample quickly dehydrates and secondly the SOD activity will be best retained at ultra low temperature. Precipitate proteins after centrifugal run of lyzate with acetone. Collect precipitate, evaporate acetone, reconstitute precipitate in a suitable buffer and assay it's SOD activity and protein content. This technique shall work fine.
Good luck!
Thank you for your suggestion. I have to purify SOD protein via an affinity column. I am using soluble fraction for protein purification. Is it ok to do acetone precipitation at -20 , further shall I include TCA as well.
Acetone precipitation can be done. Do not include TCA that will change the pH of the reconstituted protein precipitates and they may not bind to the affinity column. Which affinity column you intend to use?
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