Solarbio Phosphoenolpyruvate Carboxykinase (PEPCK) 活性測定試劑盒

屬性	細節
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	BC3310
尺寸	50T/48S

成分:

萃取液: 60 毫升×1. 4℃保存.

試劑一: 45 毫升×1, stored at 4℃.

試劑二: 粉末×1, stored at -20℃ and protected from light. Dissolve it in 35 使用前試劑 I 的毫升數. The reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

試劑三: 45 微升×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.

試劑四: 155 微升×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.

試劑V: 粉末×1, stored at -20℃ and protected from light. Dissolve it in 2.5 使用前的毫升蒸餾水, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

產品描述:

PEPCK (歐共體 4.1.1.32) 廣泛存在於動物體內, 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 奈米, which can reflect the PEPCK activity.

需要但未提供的試劑和設備

分光光度計, 低溫離心機, water bath pot, 1 ML石英比色卷, 可調式移液器, 研缽/均質器, 冰和蒸餾水

程式

我. 粗酵素液的萃取:

1. 1. 組織樣本:

組織品質比例 (G): volume of extract solution (毫升): 1:5〜10 (建議權衡一下 0.1 克組織, 添加 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 ×g 為 10 4℃ 分鐘, 取上清液置於冰上進行測試.

2. 細菌或培養細胞:

第一的, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. 細胞數量 (104): the volume of the extract (毫升) 是 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 百萬細菌或細胞), 冰浴中超音波破碎細胞 (力量: 200W or 20%, 超音波:3s, 間隔:10s, 重複 30 次). Then centrifuged at 8000 ×g 為 10 4℃ 分鐘, 取上清液置於冰上進行測試.

3. 血清樣本: 直接測定.

二. 測試 程式:

1. 將分光光度計預熱超過 30 分分鐘, 調整波長至 340 奈米, and adjust to zero with distilled
2. 工作溶液: mix Reagent II, 試劑三, Reagent IV in the proportion of 7:1:1(V:V:V) 使用前. 準備何時使用溶液.
3. Preheat the working solution at 37℃ (哺乳動物) 或者 25 ℃ (其他物種) 為了 5 分分鐘.
4. 手術台: Add the following reagents to the 1 mL quartz cuvette in turn:

試劑名稱 (微升)	空白管(乙)	試管(時間)
樣本	–	50
蒸餾水	50
工作溶液	900	900
試劑V	50	50
Add Reagent V and mix it immediately. 測量吸光度值A1 AT 340 nm for 10s and A2 at 70s. 計算ΔAT= A1T- A2T, ΔAB= A1b- A2B, and ΔA =ΔAT-ΔAB. Blank tube only needs to be done once or twice.

三、. 計算 PEPCK:

1. Calculation by micro quartz cuvette

• 以組織蛋白濃度計算:

酵素活性的定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, 每毫克蛋白質.

PEPCK activity (U/毫克蛋白質) = ΔA÷(ε×d)×VRT×109 ÷ (VS×心肺復甦) ÷ T =3215.4×ΔA÷Cpr

• 根據組織樣本的品質計算:

酵素活性的定義: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, 每克樣品.

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

• 按細胞計數:

酵素活性的定義: 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細胞)=ΔA÷(ε×d)×VRV×109÷(VS÷VST×500)÷T=6.43×ΔA

• 以血清體積計算:

酵素活性的定義: 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 (單位/毫升) = ΔA÷(ε×d)×VRV×109÷VS÷T=3215.4×ΔA

e: Molar extinction coefficient of NADH, 6.22×103公升/摩爾/厘米; d: 比色皿光徑, 1 公分;

虛擬實境測試: 反應體系總體積, 0.001 L;

VS: 反應體系中樣品體積, 0.05 毫升; 變速: 萃取液體積, 1 毫升;

心肺復甦: 樣品蛋白濃度, 毫克/毫升, Self-determination of protein concentration; 瓦: 樣品質量的質量, G;

時間: 反應時間, 1 分分鐘;

500: 細菌或細胞總數, 5 百萬; 109: Unit conversion factor, 1 mol = 109 納摩爾.

筆記:

1. 當A1小於 1 或ΔA大於 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. 在正常條件下, the change does not exceed06.
4. The steps of sample adding and mixing shall be rapid, and the stopwatch timing shall be accurate. 如果條件允許, it is recommended that two persons cooperate to complete this

實驗示例:

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/克質量) = 3215.4×ΔA÷W×10 (稀釋比例) = 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/克質量) = 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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