I am working on enzymes in Mangrove plant. Different research papers show different types of procedure. I need easy one. Also,
*Storage methods
*How I'll confirm that extraction is 100% OK for my further estimations.
*How much amount of extraction is required for determination of one enzyme.
You can follow any general protocol for extracting the crude enzyme (ensure that extraction buffer has EDTA, PVP or PVPP and mercaptoethanol or cysteine or dithiothreitol). You may download one of our papers - Prasad et al. 1999 (Prasad, K.V.S.K., Pardha Saradhi, P. and Sharmila, P. 1999. Correlative changes in the activities of antioxidant enzymes under zinc toxicity in Brassica juncea. Environ. Exptl. Bot. 42: 1-10) from my profile for getting details regarding the extraction buffer, assay buffers, substrates etc. that are required for determining activities of various antioxidants (both enzymatic and non-enzymatic).
I agree with protocol provided by Prof. P.Pardha-Saradhi. You can try the following buffer:
0.1 M phosphate buffer (pH 7.0) containing EDTA, PVP, 10% glycerol and PMSF (protease inhibitor). The homogenization part is important as the enzymes gets extracted in the buffer during the process. Perform the extraction part at 4 degree Celsius or in a cold room.
You can follow the methods described by different workers of our lab for extraction of various enzymatic and non-enzymatic antioxidants. Try to go through the following papers:
Mishra et al 2013 (Indian Journal of Biochemistry and Biophysics), Mishra and Agrawal 2014 (Protoplasma), Choudhary et al 2013 (Environmental Monitoring and Assessment) or visit https://www.researchgate.net/profile/Amit_Mishra36 for papers.
I prefer the following procedure for crude leaf extracts to determine the activities of antioxidant enzymes.
1. Approximately 200 mg (2gm) of leaf tissue was weighed and ground to a fine powder in liquid nitrogen using a pre-cooled mortar and pestle.
2. The exact weight of each powdered sample was determined before it was thoroughly homogenized in 1.2 mL of 0.2 M potassium phosphate buffer (pH 7.8 with 0.1 mM EDTA).
3. The samples were centrifuged at 15,000 xg for 20 mM at 4°C.
4. The supernatant was removed, the pellet resuspended in 0.8 mL of the same buffer, and the suspension centrifuged for another 15 min at 15,000 xg.
The combined supernatants were stored on ice and used to determine different antioxidant enzyme activities.
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