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: prah × 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, niskotemperaturna centrifuga, water bath pot, 1 ML kvarcna kiveta, Podesiva pipeta, minobacač/homogenizator, led i destilirana voda

Postupak

Ja. Ekstrakcija otopine sirovog enzima:

1. 1. Uzorak tkiva:

Udio mase tkiva (g): volume of extract solution (ml): 1:5~ 10 (preporuča se izvagati oko 0.1 g tkiva, dodati 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 × g za 10 minuta na 4 ℃, uzmite supernatant i stavite ga na led za ispitivanje.

2. Bacteria or cultured cells:

First, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. Broj ćelija (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), a stanice se razbijaju ultrazvučnim valovima u ledenoj kupelji (Vlast: 200W or 20%, ultrazvučni:3s, interval:10s, Ponoviti 30 vrijeme). Then centrifuged at 8000 × g za 10 minuta na 4 ℃, uzmite supernatant i stavite ga na led za ispitivanje.

3. Uzorak seruma: Izravno određivanje.

Ii. Test postupak:

1. Zagrijte spektrofotometar više od 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. Pripremite kada će se otopina koristiti.
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. Izračun od PEPCK:

1. Calculation by micro quartz cuvette

• Izračunato koncentracijom proteina u tkivu:

Definicija aktivnosti enzima: 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

• Izračunato prema kvaliteti uzoraka tkiva:

Definicija aktivnosti enzima: 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

• Po broju stanica:

Definicija aktivnosti enzima: 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

• Izračunato prema volumenu seruma:

Definicija aktivnosti enzima: 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: Svjetlosni promjer kivete, 1 cm;

VRT: Ukupni volumen reakcijskog sustava, 0.001 L;

Vs: Volumen uzorka u reakcijskom sustavu, 0.05 ml; Vst: Volumen otopine ekstrakta, 1 ml;

CPR: Koncentracija proteina uzorka, mg/ml, Self-determination of protein concentration; W: Masa mase uzorka, 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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