 | In vivo Nitrate Reductase Assay
1. Withdraw 3ml of cultures into test tubes
2. Remove culture medium
3. Rinse with 1ml of 1mM CaSO4 (0?C)
4. Rinse with 1ml 0f ddH2O
5. Add 2ml of reaction solution (composed of 0.1M K-buffer, pH 7.5, 0.1M KNO3,
1% n-propanol)
6. Shake (60rpm) at 30?C for 1hr in the dark
7. Bath at boiling water for 2min
8. Withdraw 0.5ml of solution into new test tube
9. Determine nitrite by the addition of 1ml of sulphanilamide reagent (1% in 2N HCl) and 1ml of N-(1-naphthyl)ethylene diamine dihydrochloride (NED) reagent (0.1% in ddH2O)
10. Develop for 30 min
11. Read at 540nm
12. Activity is expressed as ?mol nitrite/g fresh weight
References:
1. R. K. Chopra (1983) Effects of temperature on the in vivo assay of nitrate reductase in some C3 and C4 species. Ann. Bot. 51, 617-620
2. L. S. Privalle et al. (1989) Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures. Plant Physiol. 90, 962-967
3. C. S. Vogel and J. O. Dawson (1991) Nitrate reductase activity, nitrogenase activity, and photosynthesis of black alder exposed to chilling temperature. Physiol. Plant. 82, 551-558
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