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

Attribut	Détails
Note	Prenez deux ou trois échantillons différents pour la prédiction avant le test
Instrument de détection	Spectrophotomètre
Chat non	BC3400
Taille	50T/48S

Composants:

Extraire la solution: 55mL × 1, stored at 4 °C.

Réactif 1: 40mL × 1, stored at 4 °C and protected from light.

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

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

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

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

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

Description du produit:

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.

Required material

Low temperature centrifuge, spectrophotomètre, water bath/constant temperature incubator, mortier/homogénéisateur, 1 CUVETTE ML, pipette de transfert, glace et eau distillée, Tube EP.

Procédure:

je. Échantillon Extraction:

1. 1. Échantillon de tissu:

According to the mass of the tissue (g): the volume of the extract solution (ml) est 1: 5 ~ 10. Suggested 0.1g of tissue with 1mL of extract solution. Fully grind on ice, centrifugated at 20000g and 4℃ for 15 min. Supernatant is placed on ice for test.

2. Bactéries ou cellules:

According to the number of cells (104): the volume of the extract solution (ml) est 500 ~ 1000: 1. Recommend 5 million with 1mL of Extract Solution. Use ultrasonication to split bacteria or cells (power 300W, temps de travail 3s, intervalle 7s, temps total 3 min). Centrifugated at, 20000g and 4℃ for 15 min. Supernatant is placed on ice for test.

3. Liquids: direct detection.

II. Détermination procédure:

• Préchauffer le spectrophotomètre 30 min, ajuster la longueur d'onde à 340 nm, mettre à zéro avec distillé
• Ajoutez des réactifs avec la liste suivante:

Nom du réactif (µL)	Tube à essai (T)	Tube vierge (T)
Réactif 1	670	670
Réactif 2	100	100
Réactif 3	100	100
Réactif 4	10	10
Réactif 5	10	10
Réactif 6	10	10
Échantillon	100	–
Eau distillée	–	100
Après un mélange complet, measure the initial value A1 at 340 nm and the absorbance A2 for 30 minutes à 37 °C in a 1 CUVETTE ML, and record them as A1T, A1b, and A2T, A2B. Calculate ΔA = (A1T-A2T)- (A1B-A2B). Note: Réactifs 1, 2, 3, 4, 5, et 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 Calculated by micro quartz cuvette

• Calculated by protein concentration:

Définition de l'unité: 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

• Calculated by sample weight

Définition de l'unité: One unit of enzyme activity is defined as that 1g of tissue per minute consumes 1 nmol of NADH.

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

• Calculated by bacteria or cell amount:

Définition de l'unité: One unit of enzyme activity is defined as that 10 thousand bacteria or cells per minute consumes 1 nmol of NADH.

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

• Calculated by serum and other liquids:

Définition de l'unité: 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: Coefficient d'extinction molaire NADH, 6.22 × 103 L/mol/cm; d: cuvette light path, 1 cm;

CONTRE: added sample volume, 0.1 ml;

VE: volume of extract solution added, 1 ml; T: temps de réaction, 30 min;

Cpr: concentration en protéines de l'échantillon, mg/ml; W: masse de l'échantillon, 0.1 g;

109:conversion factor, 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.

Note:

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

Exemples expérimentaux:

1. Prendre 0.1 g of bean sprouts and add 1 mL of Extract solution for sample processing. After centrifugation to take the supernatant, procéder selon la procédure de détermination. CalculateΔA = (A1T-A2T)-(A1B-A2B)= (1.041-0.963)-0=0.078. The enzyme activity is calculated according to the sample mass.

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

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