Reagent A: Dissolve 387 mg of EDTA sodium salt in 10 ml of
1.361 g K2HPO4 (monobasic) in 50ml of
double distilled water
of Reagent A to 50 ml of Reagent B
the volume upto 100 ml with dH2O
Pre-chill before use.
Dissolve 100 mg of NADH in 56.4 ml of dH2O
Store under refrigeration.
Dissolve 1g sulphanilamide in 24.7 ml of 35.4% con. HCl
Make volume up to 100 ml with dH2O
0.77 mM NEDDH
Dissolve 20 mg NEDDH in 100 ml dH2O
1. Freeze dry fresh live plant samples in
liquid nitrogen and powder in a mortar using a pestle.
250mg of the sample into a 2ml eppendorff tube containing
1ml of pre-chilled extraction buffer.
Mildly shake the extraction mixture and centrifuge at 14000
rpm for 10 minutes.
of the supernatant
crude extract to a sterile clean test tube
containing 1.0 ml of 100 mM of extraction buffer
5. Add 1.6 ml of 30 mM potassium nitrate solution and
200 Ál of 2.5 mM NADH into the test tube.
assay mixture for 30 min under dark with mild shaking.
Immediately after incubation, stop the reaction by adding
ml of 58 mM sulphanilamide.
1 ml of 0.77 mM NEDDH in the tubes
The red color developed is read in the spectrophotometer at
Maintain a blank
using 200 Ál of dH2O instead of the enzyme
Prepare a standard curve by using potassium nitrite
standards of ÁM concentrations of 0, 20, 40, 60, 80 and 100
and using 200 Ál of these solutions instead of the enzyme
extract in the above procedure, but without incubation.