Solarbioホスホエノールピルビン酸カルボキシキナーゼ (ペプック) 活性アッセイキット

属性	詳細
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取得します
操作装置	分光光度計
カタログ番号	BC3310
サイズ	50T/48S

コンポーネント:

抽出液: 60 mL×1. 4℃で保存.

試薬I: 45 mL×1, stored at 4℃.

試薬II: 粉末×1, stored at -20℃ and protected from light. Dissolve it in 35 使用前の試薬 I mL. The reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

試薬Ⅲ: 45 μL×1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 1:120, to prepare when the solution will be used.

試薬IV: 155 μL×1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 7:250, to prepare when the solution will be used.

試薬V: パウダー×1, stored at -20℃ and protected from light. Dissolve it in 2.5 使用前に蒸留水 mL, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

製品説明:

ペプック (EC 4.1.1.32) 動物に広く見られる, flowering plants, algae, some fungi, and bacteria. The enzyme catalyzes the conversion of oxaloacetic acid to phosphoenolpyruvate, which is the first-rate limiting enzyme regulating gluconeogenesis.

PEPCK catalyzes oxaloacetic acid to form phosphoenolpyruvate and CO2, pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD+ in turn, and determine the NADH decline rate at 340 nm, which can reflect the PEPCK activity.

必要だが提供されていない試薬と装置

分光光度計, 低温遠心分離機, water bath pot, 1 mL石英キュベット, 調整可能なピペット, モルタル/ホモジナイザー, 氷と蒸留水

手順

私. 粗酵素液の抽出:

1. 1. 組織サンプル:

組織質量の割合 (g): volume of extract solution (mL): 1:5~10 (約の重さを量ることをお勧めします 0.1 組織グラム, 追加 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 ×gの場合 10 4℃で5分, 上清を採取し、試験のために氷上に置きます.

2. 細菌または培養細胞:

初め, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. セルの数 (104): the volume of the extract (mL) は 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 何百万もの細菌または細胞), 氷浴中で超音波により細胞を破壊する (力: 200W or 20%, 超音波:3s, 間隔:10s, 繰り返す 30 回). Then centrifuged at 8000 ×gの場合 10 4℃で5分, 上清を採取し、試験のために氷上に置きます.

3. 血清サンプル: 直接判定.

Ⅱ. テスト 手順:

1. 分光光度計を以上の時間予熱します。 30 分, 波長を調整して 340 nm, and adjust to zero with distilled
2. 実用的なソリューション: mix Reagent II, 試薬Ⅲ, Reagent IV in the proportion of 7:1:1(V:V:V) 使用前に. ソリューションをいつ使用するかを準備する.
3. Preheat the working solution at 37℃ (哺乳類) または 25 ℃ (他の種) のために 5 分.
4. 操作テーブル: Add the following reagents to the 1 mL quartz cuvette in turn:

試薬名 (μL)	ブランクチューブ(B)	試験管(T)
サンプル	–	50
蒸留水	50
実用的なソリューション	900	900
試薬V	50	50
Add Reagent V and mix it immediately. 吸収値A1を測定します 340 nm for 10s and A2 at 70s. Δat= a1tを計算します- A2t, ΔAB=A1B- A2B, and ΔA =ΔAT-ΔAB. Blank tube only needs to be done once or twice.

Ⅲ. の計算 ペプック:

1. Calculation by micro quartz cuvette

• 組織タンパク質濃度から計算:

酵素活性の定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, タンパク質のすべてのミリグラム.

PEPCK activity (U/mgプロット) = ΔA÷(ε×d)×VRT×109 ÷ (VS×CPR) ÷ T =3215.4×ΔA÷Cpr

• 組織サンプルの品質によって計算されます:

酵素活性の定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, サンプル1グラムごとに.

PEPCK activity (U/生重量g) = ΔA÷(ε×d)×VRT×109 ÷ (VS÷VST×W) ÷T= 3215.4×ΔA÷W

• 細胞数による:

酵素活性の定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every 10 thousand cells

PEPCK activity (U/104セル)=ΔA÷(ε×d)×VRV×109÷(VS÷VST×500)÷T=6.43×ΔA

• 血清量から計算:

酵素活性の定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every milliliter of serum

PEPCK activity (U/mL) = ΔA÷(ε×d)×VRV×109÷VS÷T=3215.4×ΔA

e: NADHのモル吸光係数, 6.22×103L/mol/cm; d: キュベットの軽い直径, 1 cm;

VRT: 反応系の総容積, 0.001 L;

VS: 反応系内のサンプル量, 0.05 mL; VST: 抽出液の量, 1 mL;

心肺蘇生法: サンプルタンパク質濃度, mg/mL, Self-determination of protein concentration; W: サンプル質量の質量, g;

T: 反応時間, 1 分;

500: 細菌または細胞の総数, 5 百万; 109: Unit conversion factor, 1 mol = 109 nmol.

注記:

1. A1がより少ない場合 1 またはΔAはより大きい 0.6, it is recommended to dilute the sample to a proper multiple before determination to improve the detection
2. For samples with high enzyme activity, such as animal liver, kidney and other tissues, it is recommended to dilute the extract to 5 times or more for
3. The blank tube is a test well for testing the quality of each reagent component. 通常の条件下, the change does not exceed06.
4. The steps of sample adding and mixing shall be rapid, and the stopwatch timing shall be accurate. If conditions permit, it is recommended that two persons cooperate to complete this

Experimental Example:

1. Take 0.1g liver and add 1 ml extract solution for homogenate. Take the supernatant and dilute it twice with the extract solution. Then operate according to the determination steps. Measure and calculate: ΔAT= A1D- A2D= 1.175-0.532=0.643, ΔAB=A1B – A2B =1.29-1.244=0.046, Δa=Δat – ΔAB= 0.643-0.046 = 0.597

PEPCK activity (U/g質量) = 3215.4×ΔA÷W×10 (希釈率) = 3215.4×0.597÷0.1×10=191959.4 U/g mass.

2. Take 0.1g aloe vera and add 1 ml extract solution for homogenization, take the supernatant and operate according to the determination steps. Measure and calculate ΔAT = A1T- A2T =1.257-1.106=0.151, ΔAB=A1B- A2B =1.29-1.244=0.046, Δa=Δat – ΔAB =0.151-0.046=0.105

PEPCK activity (U/g質量) = 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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