Hello everyone.
I want to do DNMT activity analysis by using prostate cancer cells. I want to know whether we can store the nuclear lysate for 2-3 days in -20 degree before performing activity analysis? Or we have to do activity analysis on the same day of nuclear lysate preparation. If we can store the samples, then for how long, they can be stored before doing activity analysis?
If your storage vial has arund 10-25% glycerol then probably you may store it in -20 oC without loosing much activity for a short period of time. Although it is better to store in 4oC or -80 oC in presence of glycerol and protease inhibitor.
A good rule of thumb is to store all cell extracts at -80oC.
Hi,
In my experience, Nuclear extract/ Nuclear lysate can be stored in -80 oC for about 6 months, provided, you totally avoid freeze/thaw cycles. It is ideal to keep the extract with protease inhibitor in aliquots within a minute or 2 (sooner the better) of extraction procedure in a -80 oC immediately following extraction process. Activity of methyltransferases is extremely sensitive to temperature variations and sample aliquots must be thawed on ice on the day of quantification and experiment. Also, the quantity of DNMTs in nucleus is extremely low that you might have to do a nuclear extraction of at least 10 - 20 million cells (depending on the cell line of use) for measuring activity of DNMTs.
Good luck!
Thank u all of you. I will store immediately or will do with fresh lysate.
It's better you store them at -80 degree. Although performing with fresh lystes is always recommended in kit based methods.
Hello,
I agree with Mr. Anirudh's suggestion. I would recommend you to use broad spectrum proteases and phosphatases inhibitors in your extraction buffers while performing cell-fractionation. Strictly perform all the steps at 4 deg. using ice for minimal activity of the above-mentioned agents. You can make aliquots in separate protease-free microfuge tubes which can be stored at -20 deg. for not more than a week (If -80 deg. not possible). Just make sure that you thaw it only on the day of experiment and use completely. The activity of inhibitors decreases after multiple free-thawing procedures.
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