筆記: 測試前取兩個或三個不同的樣本進行預測.
操作設備: 分光光度計
目錄號: BC0440
尺寸:50T/48S
成分:
- Ribulose 1,5-bisphosphate carboxylase/oxygenase (盧比斯科) is a key enzyme in plant photosynthesis.
- It controls carbon dioxide fixation and regulates the flow of carbon into the Calvin cycle and photorespiration cycle.
- The activity of Rubisco directly influences the photosynthetic rate.
- Rubisco catalyzes the combination of ribulose-1,5-diphosphate (RuBP) and carbon dioxide to produce 3-phosphoglycerate (PGA).
- PGA is further converted into glyceraldehyde-3-phosphate, accompanied by NADH oxidation to form NAD+.
- NADH absorbs light at 340 奈米, while NAD+ does not.
- In this kit, Rubisco activity is determined by measuring the decrease in NADH absorbance at 340 奈米.
需要但未提供的試劑和設備:
Ultraviolet spectrophotometer, 桌上型離心機, 可調式移液器, 水浴, 1 mL quartz cuvette, 研缽/均質器, 冰, 蒸餾水.
程式:
樣品製備:
- Bacteria or Cells:
- Collect bacteria or cells into a centrifuge tube and centrifuge to discard the supernatant.
- 添加 1 mL of Extract solution to 5 million bacteria or cells.
- Use ultrasonication (on ice, 20% 力量, 3 seconds on, 10 seconds off, repeated 30 次) to lyse bacteria or cells.
- 離心機在 10000 ×g 為 10 minutes at 4°C to remove insoluble materials and collect the supernatant on ice for testing.
- 組織:
- 添加 1 mL of Extract solution to 0.1 克組織 (preferably fresh plant samples) and homogenize on ice.
- 離心機在 10000 ×g 為 10 minutes at 4°C to remove insoluble materials, and collect the supernatant on ice for testing.
- Bacteria or Cells:
Determination Procedure:
- Preheat the UV spectrophotometer for 30 minutes and adjust the wavelength to 340 奈米. Zero the instrument with distilled water.
- Add the following reagents:
| 試劑 (微升) | 試管 (時間) | 空白管 (乙) |
| 樣本 | 100 | – |
| 蒸餾水 | – | 100 |
| 試劑三 | 35 | 35 |
| 試劑四 | 35 | 35 |
| 工作溶液 | 900 | 900 |
Detect the absorbance at 340 nm at the time of 20s and 5min20s, record as A1 and A2 respectively. ΔA(時間)=A2(時間)-A1(時間), ΔA(乙)= A2(乙)-A1(乙), ΔA=ΔA(時間)-ΔA(乙). Kept at 25℃ during the reaction. Blank tubes only need to be tested once or twice.
計算:
實驗範例:
Take 0.1g of plant leaves, 添加 1 mL of Extract solution for homogenization, 取上清液, 然後按照確定步驟進行操作.
Measure with micro quartz cuvette and calculate
ΔAT= AT1-AT2=1.279-1.206=0.073, ΔAB=AB1–AB2=0.834-0.823=0.011,
ΔA= ΔAT -ΔAB=0.073-0.011=0.062
Rubisco activity (U/克質量) = 344 × ΔA ÷ W =344×0.062÷0.1=213.28 U/g mass.
相關產品:
BC0310/BC0315 Coenzyme I NAD(H) 含量測定試劑盒
BC1030/BC1035 NAD Kinase (NADK) 活性測定試劑盒
BC0630/BC0635 NADH Oxidase (NOX) 活性測定試劑盒
BC1130/BC1135 NAD Malic Enzyme (NAD-ME) 活性測定試劑盒
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