Solarbio Phosphoenolpyruvate Carboxykinase (PEPCK) ชุดทดสอบกิจกรรม

ส่วนประกอบ:

สารสกัดโซลูชั่น: 60 mL ×1. การเก็บรักษาที่ 4 ℃.

รีเอเจนต์ I: 45 มล.×1, stored at 4℃.

รีเอเจนต์ II: powder×1, stored at -20℃ and protected from light. Dissolve it in 35 mL of Reagent I before use. The reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

รีเอเจนต์ III: 45 ไมโครลิตร×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.

รีเอเจนต์ IV: 155 ไมโครลิตร×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.

รีเอเจนต์ V: ผง×1, stored at -20℃ and protected from light. Dissolve it in 2.5 mL of distilled water before use, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

รายละเอียดสินค้า:

PEPCK (อีซี 4.1.1.32) พบมากในสัตว์, 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 นาโนเมตร, which can reflect the PEPCK activity.

รีเอเจนต์และอุปกรณ์ที่จำเป็นแต่ไม่ได้จัดเตรียมไว้ให้

สเปกโตรโฟโตมิเตอร์, เครื่องหมุนเหวี่ยงอุณหภูมิต่ำ, water bath pot, 1 mL quartz cuvette, ปิเปตแบบปรับได้, ปูน/โฮโมจีไนเซอร์, น้ำแข็งและน้ำกลั่น

ขั้นตอน

ฉัน. Extraction of crude enzyme solution:

1. 1. ตัวอย่างเนื้อเยื่อ:

The proportion of tissue mass (ก): volume of extract solution (มล): 1:5~10 (ขอแนะนำให้ชั่งน้ำหนักประมาณ 0.1 กรัมของเนื้อเยื่อ, เพิ่ม 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 ×กรัม สำหรับ 10 minutes at 4℃, นำส่วนลอยเหนือตะกอนมาวางบนน้ำแข็งเพื่อทำการทดสอบ.

2. Bacteria or cultured cells:

First, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. The number of cells (104): the volume of the extract (มล) เป็น 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 million bacteria or cells), and the cells are broken by ultrasonic wave in ice bath (พลัง: 200W or 20%, ultrasonic:3ส, ช่วงเวลา:10ส, Repeat 30 ครั้ง). Then centrifuged at 8000 ×กรัม สำหรับ 10 minutes at 4℃, นำส่วนลอยเหนือตะกอนมาวางบนน้ำแข็งเพื่อทำการทดสอบ.

3. Serum sample: Direct determination.

ครั้งที่สอง. Test ขั้นตอน:

1. Preheat the spectrophotometer for more than 30 นาที, ปรับความยาวคลื่นเป็น 340 นาโนเมตร, and adjust to zero with distilled
2. วิธีแก้ปัญหาการทำงาน: mix Reagent II, รีเอเจนต์ III, Reagent IV in the proportion of 7:1:1(วี:วี:วี) ก่อนใช้งาน. Prepare when the solution will be used.
3. Preheat the working solution at 37℃ (สัตว์เลี้ยงลูกด้วยนม) หรือ 25 ℃ (สายพันธุ์อื่น) สำหรับ 5 นาที.
4. Operation table: Add the following reagents to the 1 mL quartz cuvette in turn:

สาม. Calculation of PEPCK:

1. Calculation by micro quartz cuvette

• Calculated by tissue protein concentration:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, every milligram of protein.

PEPCK activity (U/มก) = ΔA÷(ε×d)×VRT×109 ÷ (VS× Cpr) ÷ T =3215.4×ΔA÷Cpr

• Calculated by the quality of tissue samples:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, every gram of sample.

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

• By cell count:

Definition of enzyme activity: 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 (เซลล์ U/104)=ΔA÷(ε×d)×VRV×109÷(VS÷VST×500)÷T=6.43×ΔA

• Calculated by serum volume:

Definition of enzyme activity: 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 (ยู/มล) = ΔA÷(ε×d)×VRV×109÷VS÷T=3215.4×ΔA

ε: Molar extinction coefficient of NADH, 6.22×103 L/mol/cm; ง: Light diameter of cuvette, 1 cm;

VRT: Total volume of reaction system, 0.001 ล;

VS: The volume of sample in reaction system, 0.05 มล; VST: The volume of extract solution, 1 มล;

ซีพีอาร์: ตัวอย่างความเข้มข้นของโปรตีน, มก./มล, Self-determination of protein concentration; ว: The mass of sample mass, ก;

ต: Reaction time, 1 นาที;

500: Total number of bacteria or cells, 5 ล้าน; 109: Unit conversion factor, 1 mol = 109 นาโนโมล.

บันทึก:

1. When A1 is less than 1 or ΔA is greater than 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. Under normal conditions, 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

ตัวอย่างการทดลอง:

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 (มวลยู/กรัม) = 3215.4×ΔA÷W×10 (อัตราส่วนการเจือจาง) = 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 (มวลยู/กรัม) - 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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