ソーラービオ リブロース 1 5-二リン酸カルボキシラーゼ/オキシゲナーゼ (ルビスコ) アッセイキット

注記: テスト前に予測のために 2 つまたは 3 つの異なるサンプルを採取します.

操作装置: 分光光度計

カタログ番号: BC0440

サイズ:50T/48S

コンポーネント:

• Ribulose 1,5-bisphosphate carboxylase/oxygenase (ルビスコ) 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.
• このキットでは, Rubisco activity is determined by measuring the decrease in NADH absorbance at 340 nm.

必要だが提供されていない試薬と装置:

紫外分光光度計, 卓上遠心分離機, 調整可能なピペット, 水浴, 1 mL石英キュベット, モルタル/ホモジナイザー, 氷, 蒸留水.

手順:

サンプルの準備:

• • Bacteria or Cells:
    • Collect bacteria or cells into a centrifuge tube and centrifuge to discard the supernatant.
    • 追加 1 抽出液 mL ～ 5 million bacteria or cells.
    • Use ultrasonication (on ice, 20% 力, 3 seconds on, 10 seconds off, repeated 30 回) to lyse bacteria or cells.
    • で遠心分離します。 10000 ×gの場合 10 minutes at 4°C to remove insoluble materials and collect the supernatant on ice for testing.
  • 組織:
    • 追加 1 抽出液 mL ～ 0.1 組織グラム (preferably fresh plant samples) and homogenize on ice.
    • で遠心分離します。 10000 ×gの場合 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:

吸光度を検出します。 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.

計算:

実験例:

Take 0.1g of plant leaves, 追加 1 mL of Extract solution for homogenization, 上清を取る, そして決定ステップに従って動作します.

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

関連製品:

BC0310/BC0315 Coenzyme I NAD(H) コンテンツアッセイキット

BC1030/BC1035 NAD Kinase (NADK) 活性アッセイキット

BC0630/BC0635 NADH Oxidase (NOX) 活性アッセイキット

BC1130/BC1135 NAD Malic Enzyme (NAD-ME) 活性アッセイキット