I would like to measure enzymatic activities (CAT, APx, SOD) of Myriophyllum spicatum, an aquatic plant species which is rich in phenolic compounds. I have a good yield for protein extraction, but I have no enzymatic activities.
I have tried different combinations of substrate/buffer/enzyme extract for those enzymes, so I assume my protein extraction buffer is wrong, or a step is destroying the integrity of those enzymes.
I have tested a range of extraction buffers from the literature on the same species (from pH6 to 7.8, with Sodium phosphate or Potassium phosphate) with 1mM EDTA, 2% PVP, 14mM DTE and 20% glycerol. I have tried with different times of centrifugation, and different times of crushing. Instead of using a mortar and pestle, I am using a fastprep with two glass beads, because I will have a lot of samples, and the mortar is not really an option.
However, I am always at 4°C in the ice.
Does anyone has any idea of what is going wrong and what I could change ?
Thanks !
Hi, usually those enzymes don't need to much to show activity. I have two hypothesis for you problem.
(1) You have to try a cold extraction keeping you samples all the time in ice.
(2) You might have to use cocktail of protease inhibitor with PMSF, EDTA and DTT to avoid digestion of your proteins.
In this paper " Photosynthetic and biochemical mechanisms of an EMS-mutagenized cowpea associated with its resistance to cowpea severe mosaic virus." We did these activities, maybe you can look and try something.
Best,
If you need anything let me know.
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