Solarbio Ribulose 1 5-bisphosphate carboxylase/oxygenase (Rubisco) Assay Kit

Note: Take two or three different samples for prediction before the test.

Operation Equipment: Spectrophotometer

Cat No: BC0440

Size:50T/48S

Components:

• 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.

Reagents and Equipment Required but Not Provided:

Ultraviolet spectrophotometer, desk centrifuge, adjustable pipette, water bath, 1 mL quartz cuvette, mortar/homogenizer, ice, distilled water.

Procedure:

Sample Preparation:

• • Bacteria or Cells:
    • Collect bacteria or cells into a centrifuge tube and centrifuge to discard the supernatant.
    • Add 1 mL of Extract solution to 5 million bacteria or cells.
    • Use ultrasonication (on ice, 20% power, 3 seconds on, 10 seconds off, repeated 30 times) to lyse bacteria or cells.
    • Centrifuge at 10000 ×g for 10 minutes at 4°C to remove insoluble materials and collect the supernatant on ice for testing.
  • Tissue:
    • Add 1 mL of Extract solution to 0.1 g of tissue (preferably fresh plant samples) and homogenize on ice.
    • Centrifuge at 10000 ×g for 10 minutes at 4°C to remove insoluble materials, and collect the supernatant on ice for testing.

Determination Procedure:

• • • • Preheat the UV spectrophotometer for 30 minutes and adjust the wavelength to 340 nm. Zero the instrument with distilled water.
      • Add the following reagents:

Detect the absorbance at 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.

Calculation:

Experimental example:

Take 0.1g of plant leaves, add 1 mL of Extract solution for homogenization, take the supernatant, and then operate according to the determination steps.

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/g mass) = 344 × ΔA ÷ W =344×0.062÷0.1=213.28 U/g mass.

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BC1030/BC1035 NAD Kinase (NADK) Activity Assay Kit

BC0630/BC0635 NADH Oxidase (NOX) Activity Assay Kit

BC1130/BC1135 NAD Malic Enzyme (NAD-ME) Activity Assay Kit