Solarbio Pyrophosphate: Fructose 6-phosphate-1 Phosphotransferase Assay Kit

Atribut	pojedinosti
Bilješka	Uzmite dva ili tri različita uzorka za predviđanje prije testa
Instrument za otkrivanje	Spektrofotometar
Mačka ne	BC3400
Veličina	50T/48s

Komponente:

Otopina ekstrakta: 55ml × 1, stored at 4 °C.

Reagens 1: 40ml × 1, stored at 4 °C and protected from light.

Reagens 2: Prah × 1, stored at -20 °C and protected from light. Just before use, dodati 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C for 1 week after dispensing to avoid repeated freeze-thaw cycles.

Reagens 3: powder × 1, stored at -20 °C and protected from light. Just before use, dodati 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C after dispensing. Prohibition of repeated freeze-thaw cycles.

Reagens 4: 91µL × 2, stored at 4 °C and protected from light. Just before use, dodati 0.209 mL of distilled water to fully dissolve. Stored at 4 °C for 1 week.

Reagens 5: Prah × 2, stored at -20 °C and protected from light. Just before use, dodati 0.3 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C for 1 week after dispensing.

Reagens 6: 60 µL × 1, stored at 4 °C and protected from light. Just before use, dodati 0.6 mL of distilled water to fully dissolve. Stored at 4 °C for 1 week.

Opis proizvoda:

Pyrophosphate: Fructose-6-phosphate-1-phosphotransferase (PFP, EC2.7.1.90) is a cytosolic enzyme that is widely present in plant tissues and catalyzes the phosphorylation of fructose-6-phosphate like phosphofructokinase. As a result, the single PEP catalytic reaction is a reversible reaction, and pyrophosphate is used instead of ATP, which plays an important role in carbon metabolism of photosynthesis.

PFP catalyzes the conversion of fructose 6-phosphate to fructose 1,6-diphosphate, which is converted to dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase, and then catalyzed by α-phosphate glycerol dehydrogenase and NADH to from Glycerol 3-diphosphate and NAD. The change in absorbance at 340 nm reflects the level of PFP activity.

Potreban materijal

Low temperature centrifuge, spectrophotometer, water bath/constant temperature incubator, minobacač/homogenizator, 1 ML kvarcna kiveta, PRIJENOS, led i destilirana voda, EP cijev.

Postupak:

Ja. Uzorak Izvlačenje:

1. 1. Uzorak tkiva:

Prema masi tkiva (g): the volume of the extract solution (ml) je 1: 5 ~ 10. Suggested 0.1g of tissue with 1mL of extract solution. Potpuno mljeti na ledu, centrifugated at 20000g and 4℃ for 15 min. Supernatant je stavljen na led za test.

2. Bakterije ili stanice:

Prema broju stanica (104): the volume of the extract solution (ml) je 500 ~ 1000: 1. Preporučiti 5 million with 1mL of Extract Solution. Koristite ultrazvuku za podjelu bakterija ili stanica (power 300W, Radno vrijeme 3s, Interval 7s, ukupno vrijeme 3 min). Centrifugated at, 20000g i 4 ℃ za 15 min. Supernatant je stavljen na led za test.

3. Liquids: direct detection.

Ii. Odlučnost postupak:

• Preheat the spectrophotometer 30 min, prilagoditi valnu duljinu na 340 NM, set zero with distilled
• Dodajte reagense sa sljedećim popisom:

Ime reagensa (μL)	Epruveta (T)	Prazna cijev (T)
Reagens 1	670	670
Reagens 2	100	100
Reagens 3	100	100
Reagens 4	10	10
Reagens 5	10	10
Reagens 6	10	10
Uzorak	100	–
Destilirana voda	–	100
After thorough mixing, measure the initial value A1 at 340 nm and the absorbance A2 for 30 minutes at 37 °C in a 1 ML kvarcna kiveta, and record them as A1T, A1B, and A2T, A2B. Calculate ΔA = (A1T-A2T)- (A1B-A2B). Bilješka: Reagents 1, 2, 3, 4, 5, i 6 can also be formulated into working fluids according to the proportions of the operation table, which is now prepared for use; The blank tubes need only be made 1–2 times.

Iii. Calculation of PFP activity:

1 Izračunana mikro kvarcnim kivetom

• Izračunana koncentracijom proteina:

Definicija jedinice: One unit of enzyme activity is defined as that 1 mg of tissue protein per minute consumes 1 nmol of NADH.

PFP activity (U/Mg Prot) =ΔA÷（ε×d）×VRT×109÷(VS × CPR) ÷T=53.59×ΔA÷Cpr

• Izračunana težinom uzorka

Definicija jedinice: One unit of enzyme activity is defined as that 1g of tissue per minute consumes 1 nmol of NADH.

PFP activity (U/g svježa težina) =ΔA÷（ε×d）×VRT×109÷(W ×VS÷VE) ÷T=53.59×ΔA÷W

• Izračunana bakterijama ili količinom stanica:

Definicija jedinice: One unit of enzyme activity is defined as that 10 thousand bacteria or cells per minute consumes 1 nmol of NADH.

PFP activity (U/104 ćelija) =ΔA÷（ε×d）×VRT×109÷(VS×N÷VE) ÷T=53.59×ΔA÷N（104）

• Calculated by serum and other liquids:

Definicija jedinice: One unit of enzyme activity is defined as that 1 mL of liquids per minute consumes 1 nmol of NADH.

PFP activity (U/ml) =ΔA÷（ε×d）×VRT×109÷VS÷T=53.59×ΔA

VRT: total volume of reaction system, 0.001 L;

e.: NADH Molarni koeficijent izumiranja, 6.22 × 103 L/mol/cm; d: cuvette light path, 1 cm;

Vs: added sample volume, 0.1 ml;

VE: volume of extract solution added, 1 ml; T: vrijeme reakcije, 30 min;

CPR: Koncentracija proteina uzorka, mg/ml; W: Uzorak mase, 0.1 g;

109:faktor pretvorbe, 1 mol = 109 nmol N: number of cell

2. Calculated by 96-well UV plate:

Modify d = 1 cm in the above formula to d-0.6 cm (the light path of a 96-well plate) for calculation.

Bilješka:

1. The number of samples should not be too large to avoid delaying the enzymatic reaction time.

Experimental examples:

1. uzeti 0.1 g of bean sprouts and add 1 mL of Extract solution for sample processing. After centrifugation to take the supernatant, proceed according to the determination procedure. CalculateΔA = (A1T-A2T)-(A1B-A2B)= = (1.041-0.963)-0=0.078. The enzyme activity is calculated according to the sample mass.

PFP activity (U/g svježa težina) =53.59×ΔA÷W=41.8 U/g fresh weight.

Povezani proizvodi:

BC0990/BC0995 Kit za ispitivanje biljnog klorofila

BC2210/BC2215 Glyceraldehyde-3-phosphate Dehydrogenase(Praznina) Kit za ispitivanje aktivnosti

BC4330/BC4335 biljni karotenoidni komplet za ispitivanje