Solarbio Phosphoenolpyruvate Carboxykinase (PEPCK) Kit za ispitivanje aktivnosti

Komponente:

Otopina ekstrakta: 60 mL ×1. Skladištenje na 4 ℃.

Reagens i: 45 ml × 1, stored at 4℃.

Reagens 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;

Reagens 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.

Reagens 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.

Reagens v: Prah × 1, stored at -20℃ and protected from light. Dissolve it in 2.5 ML destilirane vode prije upotrebe, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

Opis proizvoda:

PEPCK (EC 4.1.1.32) široko se nalazi kod životinja, 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.

Potrebni reagensi i oprema, ali nisu pruženi

Spektrofotometar, low temperature centrifuge, water bath pot, 1 ML kvarcna kiveta, Podesiva pipeta, minobacač/homogenizator, led i destilirana voda

Postupak

Ja. Extraction of crude enzyme solution:

1. 1. Uzorak tkiva:

The proportion of tissue mass (g): volume of extract solution (ml): 1:5~ 10 (it is recommended to weigh about 0.1 g tkiva, dodati 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 × g za 10 minutes at 4℃, take the supernatant and place it on ice for testing.

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 (ml) je 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 milijun bakterija ili stanica), and the cells are broken by ultrasonic wave in ice bath (Power: 200W or 20%, ultrasonic:3s, interval:10s, Ponoviti 30 vrijeme). Then centrifuged at 8000 × g za 10 minutes at 4℃, take the supernatant and place it on ice for testing.

3. Uzorak seruma: Direct determination.

Ii. Test postupak:

1. Preheat the spectrophotometer for more than 30 minuta, Podesite valnu duljinu na 340 NM, and adjust to zero with distilled
2. Radno rješenje: mix Reagent II, Reagens iii, Reagent IV in the proportion of 7:1:1(V:V:V) Prije upotrebe. Prepare when the solution will be used.
3. Preheat the working solution at 37℃ (sisavac) ili 25 ℃ (Ostale vrste) za 5 minuta.
4. Operativna tablica: Add the following reagents to the 1 mL quartz cuvette in turn:

Iii. 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, Svaki miligram proteina.

PEPCK activity (U/Mg Prot) = 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, Svaki gram uzorka.

PEPCK activity (U/g svježa težina) = 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 (U/104 ćelija)= 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 (U/ml) = ΔA÷(ε × D)×VRV×109÷VS÷T=3215.4×ΔA

e.: Molar extinction coefficient of NADH, 6.22× 103 l/mol/cm; d: Light diameter of cuvette, 1 cm;

VRT: Total volume of reaction system, 0.001 L;

Vs: The volume of sample in reaction system, 0.05 ml; Vst: The volume of extract solution, 1 ml;

CPR: Koncentracija proteina uzorka, mg/ml, Self-determination of protein concentration; W: The mass of sample mass, g;

T: Vrijeme reakcije, 1 minuta;

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

Bilješka:

1. Kad je A1 manji od 1 ili je ΔA veći od 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. U normalnim uvjetima, 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 masa) = 3215.4×ΔA÷W×10 (dilution ratio) = 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 masa) = = 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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