Solarbio piruvat karboksilaza (PC) Kit za ispitivanje aktivnosti

Piruvat karboksilaza (PC) Kit za ispitivanje aktivnosti

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar

Mačka ne: BC0730

Veličina:50T/48s

Komponente:

Otopina ekstrakta: Liquid 110 ml × 1. Skladištenje na 4 ℃.

Reagens i: Liquid 30 ml × 1. Skladištenje na 4 ℃.

Reagens II: Liquid 10 ml × 1. Skladištenje na 4 ℃.

Reagens iii: Prah × 1. Skladištenje na -20 ℃. Otopiti se sa 5 mL of distilled water, store at -20℃ after prepared.

Reagens IV: Prah × 1. Skladištenje na -20 ℃. Otopiti se sa 5 mL of distilled water, store at -20℃ after prepared.

Reagens v: Liquid 5 ml × 1. Skladištenje na 4 ℃.

Reagent VI: Liquid 15 μl × 1. Skladištenje na 4 ℃.

Reagent VI Diluent Solution: Liquid 10 ml × 1. Skladištenje na 4 ℃.

Opis proizvoda:

Pyruvate carboxylase (PC, EC 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 NM.

Potrebni reagensi i oprema, ali nisu pruženi:

Ultraljubičasti spektrofotometar, vodena kupelji, stolna centrifuga, vodena kupelji, Podesiva pipeta, 1 ML kvarcna kiveta, minobacač/homogenizator, led i destilirana voda.

Postupak:

Ja. Complex extraction:

• Collecting 0.1 g of tissue or 5 milijun stanica, dodati 1 ML otopine ekstrakta, grinding on ice with mortar/homogenizer.
• Centrifuga na 1000 × g za 10 minutes at4℃,
• Take the supernatant to other tube and centrifuge at 11000 × g za 15 minutes at4℃.
• The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
• Dodati 1 mL of Extract solution to the sediment, splitting with ultrasonic (vlast 20%, Radno vrijeme 5s, Interval 10s, ponoviti 12 vrijeme), used to detect the enzyme activity of PC and protein content.

Ii. Odlučnost postupak:

• Preheat ultraviolet spectrophotometer for 30 minuta, Podesite valnu duljinu na 340 NM, Postavite nulu s destiliranom vodom.
• Preheat Reagent I at 37℃ for 15 minuta.
• 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.
• Radno rješenje: make the solution as the volume ratio of Reagent II: Reagens iii: Reagent IV= 2:1:1, prepare the reagent when it will be used.
• Add the following reagents in 1 ML kvarcna kiveta:

Reagens (μL)	Prazna cijev (B)	Epruveta (T)
Reagens i	450	450
Radno rješenje	320	320
Reagens v	80	80
Reagent VI	100	100
Uzorak	–	50
Destilirana voda	50	–
Add the above reagents to the 1 mL quartz cuvette in order, timing after add working solution, Temeljito pomiješajte. Otkriti apsorbanciju na 340 nm at the time of 10 sekundi, record as AT1 or AB1. Then place dishes with the reaction solution in a 37℃ water bath for 2 minuta. Take it out and wipe it clean, immediately measure the absorbance at the time of 130 sekundi, 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.

Iii. Izračunavanje:

Definicija jedinice: 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/Mg Prot)= =[ΔA × vrv ÷(ε × D)× 109]÷(VS × CPR)÷T =1607×ΔA÷Cpr

 

e.: NADH Molarni koeficijent izumiranja, 6.22× 103 l/mol/cm; d: Lagani put kivete, 1 cm;

Vrpca: Ukupni volumen reakcije,1×10-3 L; Vs: Volumen uzorka (ml), 0.05 ml;

CPR: Koncentracija proteina uzorka (mg/ml); T: Vrijeme reakcije (min), 2 minuta;

109: 1 mol=109 nmol.

Bilješka:

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). While, extending the response time (5 minutes or 10 minuta) 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 (oko 1 mg/ml), the protein concentration of the Extract solution must be deducted when measuring the protein concentration of the sample.

4. Preporučuje se korištenje koncentracije proteina uzorka za izračunavanje aktivnosti enzima. Ako se uzorak svježe težine koristi za izračunavanje, Potrebno je izmjeriti aktivnost enzima citoplazmatskog ekstrakta, a zbroj aktivnosti enzima supernatanta i oborina je ukupna aktivnost enzima.
5. Reagents in this kit are sufficient to complete 50 reakcije cijevi.
6. Appendix: calculation formula of sample weight: (sample test number is50T/24S)

1) Supernatant:

Definicija jedinice: 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 (U/g težina) = =[ΔA1 × VRV ÷(ε × D)× 109]÷(W ÷ ve × vs)÷T =1607×ΔA1÷W

ΔA1: Supernatant absorbance;

e.: NADH Molarni koeficijent izumiranja, 6.22× 103 l/mol/cm; d: Lagani put kivete, 1 cm;

Vrpca: Ukupni volumen reakcije,1×10-3 L; Vs: Volumen uzorka (ml), 0.05 ml; VE: Extraction solution, 1 ml;

CPR: Koncentracija proteina uzorka (mg/ml); T: Vrijeme reakcije (min), 2 minuta;

109: 1 mol=109 nmol;

W: Težina uzorka, g.

2) Sediment:

Definicija jedinice: 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 (U/g težina) = =[ΔA2 × VRV ÷(ε × D)× 109]÷(W ÷ ve × vs)÷T =1607×ΔA2÷W

ΔA2: Sediment absorbance;

e.: NADH Molarni koeficijent izumiranja, 6.22× 103 l/mol/cm; d: Lagani put kivete, 1 cm;

Vrpca: Ukupni volumen reakcije,1×10-3 L; Vs: Volumen uzorka (ml), 0.05 ml;

VE: Sediment heavy suspension volume, 1 ml; CPR: Koncentracija proteina uzorka (mg/ml); T: Vrijeme reakcije (min), 2 minuta;

109: 1 mol=109 nmol;

W: Težina uzorka, g.

3) Total activity

Total activity is the sum of PC activity in supernatant and sediment. PC(U/g težina)=1607×ΔA1÷W+1607×ΔA2÷W.

Eksperimentalni primjer:

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 vrijeme. Zatim, measured by microquartzplate according to the determination steps, Supernatant: 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

Supernatant: the activity of PC (U/g masa) = = 1607 × Δ A1 ÷ W × 100 (dilution ratio) = 1607×0.215÷0.1× 100 = = 345505 U/g masa;

Precipitation: the enzyme activity of PC (U/g masa) = 1607×ΔA2 ÷ W×4 (dilution ratio) = 1607×0.321÷ 01.

× 4=20633.88 U/g mass;

The total enzyme activity of PC (U/g masa) = 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.

Reference

[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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