Solarbio丙酮酸羧化酶 (個人電腦) 活性測定試劑盒

丙酮酸羧化酶 (個人電腦) 活性測定試劑盒

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: BC0730

尺寸:50T/48S

成分:

萃取液: 液體 110 毫升×1. 4℃保存.

試劑一: 液體 30 毫升×1. 4℃保存.

試劑二: 液體 10 毫升×1. 4℃保存.

試劑三: 粉末×1. -20℃保存. 溶解 5 毫升蒸餾水, store at -20℃ after prepared.

試劑四: 粉末×1. -20℃保存. 溶解 5 毫升蒸餾水, store at -20℃ after prepared.

試劑V: 液體 5 毫升×1. 4℃保存.

試劑六: 液體 15 微升×1. 4℃保存.

Reagent VI Diluent Solution: 液體 10 毫升×1. 4℃保存.

產品描述:

Pyruvate carboxylase (個人電腦, 歐共體 6.4.1.1) is widely present in mitochondria of animals, molds and yeast, but is not found in plants and most bacteria. PC is the main postreaction for oxaloacetate, and is the first-rate- limiting enzyme in the gluconeogenesis process.

PC irreversibly catalyzes pyruvate, ATP, CO2 and water to oxaloacetate, ADP and Pi, malic dehydrogenase further catalyzes the formation of malic acid and NAD+ from acetoacetic acid and NADH. The enzyme activity of PC can be reflected by detecting the oxidation rate of NADH at 340 奈米.

需要但未提供的試劑和設備:

紫外分光光度計, 水浴, 桌上型離心機, 水浴, 可調式移液器, 1 ML石英比色卷, 研缽/均質器, 冰和蒸餾水.

程式:

我. Complex extraction:

• Collecting 0.1 g of tissue or 5 百萬個細胞, 添加 1 萃取液毫升數, grinding on ice with mortar/homogenizer.
• 離心機在 1000 ×g 為 10 minutes at4℃,
• Take the supernatant to other tube and centrifuge at 11000 ×g 為 15 minutes at4℃.
• The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
• 添加 1 mL of Extract solution to the sediment, splitting with ultrasonic (力量 20%, work time 5s, 間隔10秒, 重複 12 次), used to detect the enzyme activity of PC and protein content.

二. 決心 程式:

• Preheat ultraviolet spectrophotometer for 30 分分鐘, 調整波長至 340 奈米, 用蒸餾水調零.
• Preheat Reagent I at 37℃ for 15 分分鐘.
• Diluent Reagent VI according to the volume ratio of Reagent VI: Reagent VI Diluent Solution = 1.6:660(V:V), prepare the reagent when it will be used.
• 工作溶液: make the solution as the volume ratio of Reagent II: 試劑三: Reagent IV= 2:1:1, prepare the reagent when it will be used.
• 將下列試劑加入 1 ML石英比色卷:

試劑 (微升)	空白管 (乙)	試管 (時間)
試劑一	450	450
工作溶液	320	320
試劑V	80	80
試劑六	100	100
樣本	–	50
蒸餾水	50	–
Add the above reagents to the 1 mL quartz cuvette in order, timing after add working solution, 充分混合. 檢測吸光度 340 nm at the time of 10 秒, record as AT1 or AB1. Then place dishes with the reaction solution in a 37℃ water bath for 2 分分鐘. Take it out and wipe it clean, immediately measure the absorbance at the time of 130 秒, which record as AT2 or AB2. ΔAT= AT1- AT2, ΔAB=AB1- AB2, ΔA= ΔAT-ΔAB. The blank tube only need to test once or twice.

三、. 計算:

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every milligram of protein.

PC Activity(U/毫克蛋白質)=[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =1607×ΔA÷Cpr

 

e: NADH摩爾滅絕係數, 6.22×103公升/摩爾/厘米; d: 淺色小路, 1 公分;

繩索: 總反應體積,1×10-3 L; VS: 樣品量 (毫升), 0.05 毫升;

心肺復甦: 樣品蛋白濃度 (毫克/毫升); 時間: 反應時間 (分分鐘), 2 分分鐘;

109: 1 mol=109 nmol.

筆記:

1. Take one or two different samples for prediction before test. It is recommended to dilute the crude enzyme solution with the Extract solution before the determination if the ΔA>0.8(if measuring with 96 well flat-bottom UV plate, ΔA>0.5). 儘管, extending the response time (5 minutes or 10 分分鐘) if ΔA <0.01.

2. The blank tube is a detection hole for detecting the quality of each reagent component, and normally that the change of ΔAB does not exceed 0.05.
3. The protein concentration of the sample needs to be determined by yourself. Since the Extract solution contains a relatively high protein concentration (關於 1 毫克/毫升), the protein concentration of the Extract solution must be deducted when measuring the protein concentration of the sample.

4. It is recommended to use the sample protein concentration to calculate the enzyme activity. If the sample fresh weight is used to calculate, the enzyme activity of cytoplasmic extract needs to be measured, and the sum of supernatant and precipitation enzyme activity is the total enzyme activity.
5. Reagents in this kit are sufficient to complete 50 tube reactions.
6. Appendix: calculation formula of sample weight: (sample test number is50T/24S)

1) 上清液:

單位定義: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.

PC Activity (單位/克重量) =[ΔA1×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA1÷W

ΔA1: Supernatant absorbance;

e: NADH摩爾滅絕係數, 6.22×103公升/摩爾/厘米; d: 淺色小路, 1 公分;

繩索: 總反應體積,1×10-3 L; VS: 樣品量 (毫升), 0.05 毫升; 維: Extraction solution, 1 毫升;

心肺復甦: 樣品蛋白濃度 (毫克/毫升); 時間: 反應時間 (分分鐘), 2 分分鐘;

109: 1 mol=109 nmol;

瓦: 樣品重量, G.

2) Sediment:

單位定義: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.

PC Activity (單位/克重量) =[ΔA2×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA2÷W

ΔA2: Sediment absorbance;

e: NADH摩爾滅絕係數, 6.22×103公升/摩爾/厘米; d: 淺色小路, 1 公分;

繩索: 總反應體積,1×10-3 L; VS: 樣品量 (毫升), 0.05 毫升;

維: Sediment heavy suspension volume, 1 毫升; 心肺復甦: 樣品蛋白濃度 (毫克/毫升); 時間: 反應時間 (分分鐘), 2 分分鐘;

109: 1 mol=109 nmol;

瓦: 樣品重量, G.

3) 全部的 活動

Total activity is the sum of PC activity in supernatant and sediment. 個人電腦(單位/克重量)=1607×ΔA1÷W+1607×ΔA2÷W.

實驗範例:

1. 1 mL of Extract solution is added to 0.1 g of rabbit heart tissue for homogenization. The supernatant is diluted 100 times with Extract solution, and the precipitation was diluted 4 次. 然後, measured by microquartzplate according to the determination steps, 上清液: the ΔAT = A1T – A2T = 1.104-0.856 =0.248, ΔAB = A1B – A2B = 1.021-0.988=0.033, Δ A1 = ΔAT – ΔAB = 0.248-0.033=0.215, precipitate: ΔAT= A1T – A2T = 1.07-0.716 =0.354, ΔAB= A1b- A2B = 1.021-0.988=0.033, ΔA2 = ΔAT- ΔAB =0.354-0.033= 0.321

上清液: the activity of PC (U/克質量) = 1607 × Δ A1 ÷ W × 100 (稀釋比例) = 1607×0.215÷0.1× 100 = 345505 U/克質量;

沉澱: the enzyme activity of PC (U/克質量) = 1607×ΔA2 ÷ W×4 (稀釋比例) = 1607×0.321÷ 01.

× 4=20633.88 U/g mass;

The total enzyme activity of PC (U/克質量) = 1607×ΔA1÷W×100 (dilution) + 1607×ΔA2 ÷ W

=1607 × 0.215 ÷0.1×100+1607×0.321÷0.1×4=366138.88 U/g mass.

參考

[1] Esmail S. Kakey,Amez A. Ismael. Evaluation of Oxidative Stress Status in Aged Human in relation to some Diseases. International Conference on Pure and Applied Sciences. August 2018;

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