相关酶活测定方案-微量经典酶活测定11-21(1).doc

Extraction Buffer: H2O 50 mM Tris-HCl PH7.0 20% Glycerol, 1 mmol/L ASA, 1 mmol/L DTT, 1 mmol/L GSH, 5 mmol/L MgCl2 Combine 4% PVPP, and 2 ml of the extraction buffer to a pre-cooled mortar. Add 2 leaf discs (1.16 cm2) and grind with pre-cooled pestle. Centrifuge extract for 15 min at 1 Non-specific Peroxidase (POD) ? 470 reaction rate in 1 min , Test the lag 15 seconds. Use 10?l 1M PBS PH6.0 Total volume: 1 ml 20 ml Reaction solution: 1M PBS PH6.0 4ml 30%H2O2 11.2 ?l Guaiacol 7.6 ?l H2O 15.98 ml 990 ?l reaction solution Initiate reaction by adding 10 ?l of plant extract to each cuvette. Set Abs range from 0 - 1 Extinction coefficient: 26.6 mM-1 Superoxide Dismutase (SOD) ? 560 Reaction solution: 50 mM PBS PH7.0, 77.12 μM NBT, 0.1 mM EDTA, 13.37 mM Met NO1 and NO3 cuvette. 3.85 ml reaction solution 100 ?l Riboflavin Leaf 50 ?l sample (root 100 ?l ) NO2 cuvette. 3.85 ml reaction solution 100 ?l Riboflavin Leaf: 50 ?l 50mM PBS PH7.0, (root:100 ?l ) Correspondingly same volume of 50mM PBS PH7.0 instead for sample Then put NO1 and NO2 in light of 4000lx at 25℃ Put NO3 in dark at 25℃ for 30min SOD unit/ml=[V/v-1]*(dilution factor) Malondialdehyde (MDA) extraction and determination 6 leaf discs (3.48 cm2) or root 90mg~100mg were ground in liquid nitrogen with a pestle and mortar and extracted with 2.0ml (root 1.0ml) 80% ethanol, followed by centrifugation at 3000g Reation solution: No1 20% TCA 784 ?l 0.5% BHT 16 ?l (use 80% ethanol to dissolve) Sample 200 ?l No2 is the same to No1 except using 80% ethanol instead for sample No3 0.65% (w/v)TBA 784 ?l (use 20% TCA to dissolve) 0.5% BHT 16 ?l Sample 200 ?l No4 is the same to No3 except using 80% ethanol instead for sample NO1.2.3.4 cuvette mixtured vigorously, and heat at 95℃ for 25 min,cooled ,and centrifuged at 3000g A=[(Abs532+TBA