In our lab we work with a phospholipase A2 for one of our subprojects. We purified the protein and we check the activity by testing cleavage of NBD Avanti lipids followed by TLC run. Reactions were done in HEPES buffer pH 7.5 with 0.5 mM DDM, 100mM NaCl and 10 mM CaCl2. We managed to confirm PLA2 activity several times for all our aliquots in the past, using our 10 batches of purified protein (the oldest one from 10 months ago and the most recent from last week). Last confirmation of activity was 3 weeks ago.

Nevertheless, we couldn't observe activity when we tried to confirm the activity of the newest batch 3 days ago. We decided to also use the previous batches as controls, but none of them showed activity this time neither. On the other hand, a positive control of commercial PLA2 showed positive activity, confirming that the conditions should be right. We decided to test several concentrations of the elements of the buffer (DDM, NaCl, CaCl2) to Tris, different protein concentrations, different temperatures and pH, as well as with and without shaking; but we were not successful in any case.

We store our proteins at -80ºC in 50% glycerol and in aliquots to prevent thawing/unthawing cycles. Therefore, I would not think that all the batches died because of time/storing conditions.

Any help would be greatly appreciated. Thanks in advance!