Nitrate Reductase (NR) Colorimetric Assay Kit

Transcription

1 (FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGNOSIS!) Nitrate Reductase (NR) Colorimetric Assay Kit Catalog No: E-BC-K158 Method: Colorimetric method Specification: 100 Assays This manual must be read attentively and completely before using this product. If you have any problems, please contact our Technical Service Center for help. Phone: (USA) Fax: (USA) Website: Please kindly provide us the lot number (on the outside of the box) of the kit for more efficient service. Copyright Elabscience Biotechnology Inc. All Rights Reserved

2 Application This kit can be used for measurement of NR activity fresh plant and other samples. This kit (100 Assays) can detect 48 samples. Detection significance NR is a key enzyme of nitrogen assimilation in plant nitrogen metabolism. It is related with nitrogen absorption in crop, and can affect the yield and quality of crop. Therefore, the NR activity can be considered as one of the indexes of plant nutrition or fertilization, and it can also be used as an important indicator of varieties breeding. Detection principle NR can catalyze the reduction of nitrate in plants and form nitrite. The produced nitrite then reacts with chromogenic agent and form red compounds, which can be colorimetric assayed stably at 540 nm. Kits components Specification Storage Reagent 1 2 mol/l NR standard stock solution, 1 ml 1 vial 4, 3 months Preparation of 100 μmol/ml NR standard application solution: Dilute the NR standard stock solution for 20 times with double distilled water (1:19). Reagent 2 Substrate, 100 ml 1 vial 4, 3 months Reagent 3 Chromogenic agent, 100 ml 1 vial 4, 3 months Reagent 4 Homogenate medium, 100 ml 1 vial 4, 3 months Reagent 5 Revulsive stock solution, 100 ml 1 vial 4, 3 months Preparation of Revulsive application solution: Dilute the Revulsive stock solution with double distilled water for 4 times (1:3) before use. Experimental instrument Test tube, Micropipettor, Vortex mixer, 37 water bath, Low-speed centrifuge, Spectrophotometer (540 nm). Sample pretreatment 1. Add the Reagent 5 (inducer application solution) into a beaker. 2. Take fresh plant sample, wash and dry with filter paper. Take the whole or part of the plant tissue into the inducer application solution, immerse and induce for 2 hours. 3. Take the induced sample and dry it with filter paper, then freeze at -20 for 30 min. Dry with filter paper and weigh the sample. 4. Add the Reagent 4 (homogenate medium) in a Weight (g): Volume (ml) ratio of 1: 9, make mechanical homogenization in ice water bath to prepare 10% tissue homogenate. Centrifuge at 3500 rpm for 10 min, then take the supernatant for detection. 2

3 Operation steps Blank Standard Sample Control tube tube tube tube Double-distilled water (ml) μmol/l standard application solution (ml) 0.2 Sample (ml) 0.2 Reagent 2, substrate (ml) Mix fully and incubate in 37 water bath in dark for 30 min. Reagent 3, chromogenic agent (ml) Sample (ml) 0.2 Mix fully. Centrifuge at 3500 rpm for 10 min. Take the supernatant and measure the OD values of each tube at 540 nm wavelength with 1cm diameter cuvette and set to zero with double-distilled water. Calculation of results NR activity (U/mg) = OD Sample OD Control OD Standard OD Blank Concentration of standard (100 μmol/l) Sampling volume (0.2 ml) Wet weight of sample (g) Reaction time (30 min) Dilution factor of sample before tested Notes 1. NR is easy to be reduced, so the detection should be operated quick and be controlled under 4 condition. 2. Sampling is recommended to be operated in fine weather to ensure the enough illumination of plant. Or apply a certain amount of nitrate nitrogenous fertilizer, which can increase the enzyme activity. The sampling region must be keep coincident. 3. The enzyme reaction should be protected from light to avoid of formation of reduced ferredoxin in chloroplast under illumination condition. 4. The color developing reaction of this kit is stable. The colorimetric assay can be finished with 1 hour. 5. This kit is for research use only. 3

4 Preparation of Nitrate reductase Standard Curve Pretreatment Dilute the 2 mmol/lnr standard stock solution into different concentrations (0 μmol/l, 25 μmol/l, 50 μmol/l, 100 μmol/l, 200 μmol/l, 400 μmol/l, 800 μmol/l) with the Double distilled water. Operation steps Blank tube Standard tube Double-distilled water (ml) 0.2 Standard solution (ml) 0.2 Reagent 2, substrate (ml) 1 1 Mix fully and incubate in 37 water bath in dark for 30 min. Reagent 3, chromogenic agent (ml) 1 1 Detection results Concentration of standard (μmol/l) Measured OD Absolute OD

5 Standard curve Copyright Elabscience Biotechnology Inc. All Rights Reserved 5