Plant Rubisco Activity Assay Kit Instructions

Plant Rubisco Activity Assay Kit

● Product introduction:

Ribisco-1,5-bisphosphate carboxylase (Rubisco) is an important regulatory enzyme in photosynthesis carbon metabolism. It is the most abundant protein in plants, mainly in the soluble part of chloroplasts, and the total amount is about chloroplast. Soluble protein 50% to 60%.

Here, one molecule of CO ₂ is fixed, and two molecules of NADH are oxidized. Therefore, the activity of Rubisco can be calculated from the amount of oxidation of NADH, and the amount of NADH oxidation can be calculated from the change in absorbance at 340 nm.

In order to synchronize the oxidation of NADH with the fixation of CO ₂ , it is necessary to add an ATP regeneration system of creatine phosphate (CrP) and phosphocreatine kinase (CPK).

The kit can measure 200 samples using a 0.5 ml reagent 1-Rubisco extraction buffer (2×).

● Bring your own materials:

Plant material; scissors; liquid nitrogen; mortar or other grinding equipment; 1.5 ml centrifuge tube; 1 ml quartz cuvette; ultraviolet spectrophotometer; ice machine;

● Operation steps:

First, ready-to-use Rubisco extraction buffer preparation:

Note: 1. The ready-to-use Rubisco extraction buffer should be used as soon as possible, and can be stored overnight at 4 °C .

2. To enhance the stability of Rubisco , protease inhibitors can be added to the extraction buffer ( Cat : PC2030 )

Second , Rubisco sample extraction:

1. Take fresh plant tissue, clean it, dry it, grind it in liquid nitrogen, add 1 cm ² leaf (1 cent coin size) or 0.1 g powder to 1 ml ready-to-use Rubisco extraction buffer, mix by inversion, place in ice bath. 5-10 minutes.

After centrifugation at 2.12000g at 4°C for 10 minutes, the supernatant is Rubisco sample extract, which is placed at room temperature for standby (do not place in ice bath or 4 °C, and keep Rubisco activity at room temperature).

Note: Rubisco sample extracts are best measured immediately and are not recommended for storage.

Third , Rubisco activity determination analysis :

1. Ready-to-use enzyme lysis buffer preparation:

Add 1.5 ml of sterilized water and 1 ml of BSA solution (50 mg/ml) to the reagent 10-enzyme lysis buffer original bottle as shown on the label. Mix well and prepare a ready-to-use enzyme lysis buffer. , stored at -20 °C.

2. Reagent 3-GAPDH and reagent 5-CPK were added to the ready-to-use enzyme lysis buffer as indicated on the label, mixed and dissolved, and then stored in an ice bath at -20 °C.

3. Reagent 4-PGK is ammonium sulfate suspension, centrifuge at 12000g for 5 minutes at 4°C, remove the supernatant (carefully remove, keep a little supernatant, do not absorb the precipitate), add the ready-to-use enzyme to dissolve in the precipitate as indicated on the label. Buffer, mix and dissolve, then rinse in ice bath, store at -20 °C.

4. Reagent 9-RuBP: Add sterilized water as indicated on the label, mix thoroughly and then rinse in ice bath.

Note: The stability of RuBP solution is poor. It should be stored at -20 °C after use, valid for 3-4 days, and stored at -80 °C for a long time.

5. Reagent 8-NADH: Add sterilized water as indicated on the label, mix thoroughly and then rinse in ice bath.

Note: NADH has poor stability. It should be stored at -20 °C for 3-4 days and stored at -80 °C for a long time.

6. Reaction system MasterMix preparation:

7. Determination of the reaction system;

The following reaction was prepared in a 1.5 ml centrifuge tube.

Note: The general spectrophotometer OD ₃₄₀ reading is accurate between 0.5-3. Below this range, the material used for extraction is too small, and the Rubisco activity is too low. Above this range, the material used is too much, and the Rubisco activity is too high. At this time, the Rubisco sample extract can be diluted with a ready-to-use Rubisco extraction buffer and then measured.

Fourth , Rubisco activity calculation:

Calculate the initial and total activity of Rubisco in the sample according to the following formula:

Activity (μmol·m ^-2 ·S ^-1 ) - indicates how many μmol of NADH is oxidized per square meter of blade per second

The absolute value of the tube absorbance change at 340 nm in the first 1 minute of the ΔA-reaction

N-dilution factor, 40 in this procedure (1 ml extract, determined by 25 μl).

6.22 - Absorbance coefficient at 340 nm per micromolar NADH

D-cm cuvette optical path, generally 1

â–³ t-second measurement time, the program is 60 seconds

Leaf area used for S-cm ² extraction

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