Nota: Prendi due o tre campioni diversi per la previsione prima del test.
Attrezzatura operativa: Spettrofotometro
Gatto n: BC0440
Misurare:50T/48S
Componenti:
- Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) 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 nm, while NAD+ does not.
- In this kit, Rubisco activity is determined by measuring the decrease in NADH absorbance at 340 nm.
Reagenti e attrezzature necessari ma non forniti:
Spettrofotometro ultravioletto, centrifuga da tavolo, pipetta regolabile, bagnomaria, 1 Cuvetta in quarzo da ml, mortaio/omogeneizzatore, ghiaccio, acqua distillata.
Procedura:
Preparazione del campione:
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- Bacteria or Cells:
- Collect bacteria or cells into a centrifuge tube and centrifuge to discard the supernatant.
- Aggiungere 1 mL of Extract solution to 5 million bacteria or cells.
- Use ultrasonication (on ice, 20% energia, 3 seconds on, 10 seconds off, repeated 30 volte) to lyse bacteria or cells.
- Centrifugare a 10000 ×g per 10 minutes at 4°C to remove insoluble materials and collect the supernatant on ice for testing.
- Tessuto:
- Aggiungere 1 mL of Extract solution to 0.1 g di tessuto (preferably fresh plant samples) and homogenize on ice.
- Centrifugare a 10000 ×g per 10 minutes at 4°C to remove insoluble materials, and collect the supernatant on ice for testing.
- Bacteria or Cells:
Determination Procedure:
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-
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- Preheat the UV spectrophotometer for 30 minutes and adjust the wavelength to 340 nm. Zero the instrument with distilled water.
- Add the following reagents:
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| Reagente (μL) | Provetta (T) | Tubo vuoto (B) |
| Campione | 100 | – |
| acqua distillata | – | 100 |
| Reagente III | 35 | 35 |
| Reagente IV | 35 | 35 |
| Soluzione funzionante | 900 | 900 |
Rilevare l'assorbanza a 340 nm at the time of 20s and 5min20s, record as A1 and A2 respectively. ΔA(T)=A2(T)-A1(T), ΔA(B)= A2(B)-A1(B), ΔA=ΔA(T)-ΔA(B). Kept at 25℃ during the reaction. Blank tubes only need to be tested once or twice.
Calcolo:
Esempio sperimentale:
Take 0.1g of plant leaves, aggiungere 1 mL of Extract solution for homogenization, prendi il surnatante, e quindi operare secondo le fasi di determinazione.
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 (Massa U/g) = 344 × ΔA ÷ W =344×0.062÷0.1=213.28 U/g mass.
Prodotti correlati:
BC0310/BC0315 Coenzyme I NAD(H) Kit di analisi del contenuto
BC1030/BC1035 NAD Kinase (NADK) Kit di analisi dell'attività
BC0630/BC0635 NADH Oxidase (NOX) Kit di analisi dell'attività
BC1130/BC1135 NAD Malic Enzyme (NAD-ME) Kit di analisi dell'attività
Recensioni
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