Phosphofructokinase (PFK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: P1501170

QUALITÉ & PURETÉ BioReagent

Vue d’ensemble

Phosphofructokinase (PFK, EC 2.7.1.11) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the conversion of fructose-6-phosphate and ATP to fructose-1,6-bisphosphate and ADP, serving as one of the key regulatory enzymes in the glycolysis process.

Detection Principle: PFK catalyzes the conversion of fructose-6-phosphate and ATP to fructose-1,6-bisphosphate and ADP. Pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to generate NAD⁺. The PFK activity is reflected by measuring the rate of decrease in NADH at 340 nm.

Applicable Samples: Animal/plant tissues, cells, cell supernatant, bacteria, serum (plasma).

P1501170	Component	48T	96T	Storage
P1501170A	Extraction Buffer	60 mL	60 mL×2	2-8℃
P1501170B	Assay Buffer	11 mL	22 mL	2-8℃
P1501170C	Substrate Mix	1EA	1EA	2-8℃. Store in the dark.
P1501170D	Enzyme 1	600 μL	1.2 mL	-20℃. Store in the dark.
P1501170E	Enzyme 2	600 μL	1.2 mL	-20℃. Store in the dark.

Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.

User-Provided Instruments and Reagents

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 340 nm.
Consumables	96-well Microplate	UV-transparent plate.
Reagents	PBS (pH 7.4)	For washing samples.
Others	Homogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tips	Using a multichannel pipette for large-scale detection can improve efficiency.

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Working Substrate Mix	Prepare before use: For 48T: Add 10.2 mL Assay Buffer and 0.678 mL deionized water to the vial. For 96T: Add 20.4 mL Assay Buffer and 1.356 mL deionized water to the vial. Dissolve thoroughly.	Protect from light during the experiment. Unused Working Substrate Mix can be aliquoted and stored at -20°C protected from light for 2 weeks. Avoid repeated freeze-thaw cycles.
Enzyme 1	Ready-to-use; equilibrate to room temperature before use.	Protect from light during the experiment; store at -20°C protected from light.
Enzyme 2	Ready-to-use; equilibrate to room temperature before use.	Protect from light during the experiment; store at -20°C protected from light.

2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.

2.1 Animal Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant into a new tube and keep on ice for detection.
2.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.3 Cells or Bacteria: Collect 5×10⁶ cells or bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.4 Serum (Plasma), Cell Supernatant: Detect directly.

3. Assay Steps
3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 340 nm.
3.2 Assay System Setup: In a 96-well UV plate, add sequentially: 10 µL of sample, 10 µL of Enzyme 1, 10 µL of Enzyme 2, and 170 µL of Working Substrate Mix. Mix well.
3.3 Absorbance Measurement: Immediately record the absorbance at 340 nm at 20 seconds (A1) and then again after 10 minutes and 20 seconds (A2). Calculate ΔA = A1 - A2.

4. Result Calculation
The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.

4.1 Data Processing
Calculate ΔA = A1 - A2.

4.2 Sample PFK Activity Calculation

(1) Based on sample mass: 

 Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per gram of tissue in the reaction system. 

 Formula: 

PFK (U/g fresh weight) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (V _sample ÷ V _{total extract} × W) ÷ T = 643 × ΔA ÷ W 

(2) Based on cell or bacterial count: 

 Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per 10⁴ cells or bacteria in the reaction system. 

 Formula: 

PFK (U/10⁴) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (V _sample ÷ V _{total extract} × N) ÷ T = 643 × ΔA ÷ N 

(3) Based on liquid volume: 

 Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per mL of serum (plasma) or cell supernatant in the reaction system. 

 Formula: 

PFK (U/mL) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ V _sample ÷ T = 643 × ΔA 

(4) Based on protein concentration: 

 Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per mg of protein in the reaction system. 

 Formula: 

PFK (U/mg prot) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (Cpr × V _sample ) ÷ T = 643 × ΔA ÷ Cpr 

 Parameter Description: 

 V _{total reaction} : Total reaction volume, 2.0 × 10⁻⁴ L 

 ε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cm 

 d: Light path of the 96-well plate, 0.5 cm 

 10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol) 

 V _sample : Volume of sample added, 0.01 mL 

 V _{total extract} : Volume of Extraction Buffer added, 1 mL 

 W: Sample mass, g 

 T: Reaction time, 10 min 

 Cpr: Sample protein concentration, mg/mL 

 N: Cell or bacterial count, in units of 10⁴ (e.g., for 5×10⁶ cells, N=500)

5. Result Presentation
Example: 0.1 g of corn seeds was processed and assayed according to the procedure using a 96-well UV plate.
Measured: ΔA = A1 - A2 = 0.6772 - 0.6131 = 0.0641.
Calculated based on sample mass:
PFK (U/g fresh weight) = 643 × ΔA ÷ W = 411.5 U/g fresh weight.

Precautions

1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

3. For tissue and cell samples, results can be normalized by measuring the protein concentration. Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.

5. This product is for scientific research use only. Not intended for clinical diagnosis.

Frequently Asked Questions
Q: What should I do if the sample ΔA is too high or too low?
A: If the sample ΔA is > 0.5, the PFK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer or reduce the amount of sample used for extraction, and re-assay. If the sample ΔA is < 0.002, increase the sample amount.

Spécifications

Synonymes

EC2.7.1.11

Spécifications et pureté

BioReagent

Grade

BioReagent

Application

Cell Metabolism, Enzyme activity assay

Sensibilité

Light-sensitive

Stockage et expédition

Conditions de stockage de stockage

Store at 2-8°C,Protected from light,Store at -20°C

Expédié en

Ice chest + Ice pads

Stabilité et stockage

Each component has a shelf life of 1 year under corresponding storage conditions.