Pyruvate Kinase (PK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: A1501205

GRADE & PURITY BioReagent

Overview

Pyruvate Kinase (PK, EC 2.7.1.40) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis and is one of the key rate-limiting enzymes in glycolysis, as well as a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.

Detection Principle: PK catalyzes the conversion of phosphoenolpyruvate and ADP to ATP and pyruvate. Lactate dehydrogenase (LDH) further catalyzes the reaction of NADH and pyruvate to produce lactate and NAD⁺. The change in NADH absorbance at 340 nm is measured to calculate the PK activity in the sample.

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

A1501205	Component	48T	96T	Storage
A1501205A	Extraction Buffer	60 mL	60 mL×2	2-8℃
A1501205B	Assay Buffer	12 mL	24 mL	2-8℃
A1501205C	Substrate Mix	1EA	1EA	-20℃. Store in the dark.
A1501205D	LDH	10.2 μL	20.4 μL	2-8℃

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 UV Plate	UV-transparent plate.
Reagents	PBS (pH 7.4) / Deionized Water	For washing cells/bacteria / Reagent preparation.
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.
Substrate Mix Working Reagent	Prepare before use: For 48T: Add 10.2 mL Assay Buffer and 0.6 mL deionized water to the vial. For 96T: Add 20.4 mL Assay Buffer and 1.2 mL deionized water to the vial. Dissolve thoroughly.	After preparation, aliquot and store at -20°C for up to 6 months. Avoid repeated freeze-thaw cycles. Before use, incubate at 25°C (for general species) or 37°C (for mammals) for 5 min.
LDH Working Reagent	Prepare before use: For 48T: Add 0.6 mL deionized water to the LDH vial. For 96T: Add 1.2 mL deionized water to the LDH vial. Mix thoroughly.	Keep on ice after preparation. The diluted reagent can be stored at 4°C for 1 month.

2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.

2.1 Animal/Plant 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 and keep on ice for detection.
2.2 Cells/Bacteria: Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, 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.
2.3 Serum (Plasma) and other liquid samples: 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 LDH Working Reagent, and 180 µL of Substrate Mix Working Reagent. Mix rapidly immediately after addition.
3.3 Absorbance Measurement: Immediately after mixing, measure the absorbance at 340 nm at 20 seconds (A1) and then at 2 minutes and 20 seconds (A2). Calculate ΔA = A1 - A2.

4. Result Calculation

4.1 Data Processing
Calculate ΔA = A1 - A2.

4.2 Sample PK Activity Calculation

(1) Based on sample mass:

• Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per gram of tissue in the reaction system.

• Formula:

• PK (U/g) = [ΔA×V_{total reaction}÷ (ε × d) × 10⁹] ÷ (V_sample ÷ V_{total extract}×W) ÷ T = 3215 × ΔA ÷ W

(2) Based on cell/bacterial count:

• Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per 10⁴ bacteria or cells in the reaction system.

• Formula:
  PK (U/10⁴) = [ΔA × V_{total reaction} ÷ (ε × d) × 10⁹] ÷ (V_sample ÷ V_{total extract}; × 500) ÷ T = 3215 × ΔA ÷ 500 = 6.431 × ΔA

(3) Based on liquid volume:

• Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mL of serum (plasma) in the reaction system.

• Formula:
  PK (U/mL) = [ΔA × V_{total reaction}; ÷ (ε × d) × 10⁹] ÷ V_sample ÷ T = 3215 × ΔA

(4) Based on protein concentration:

• Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mg of protein in the reaction system.

• Formula:
  PK (U/mg prot) = [ΔA × V_{total reaction}; ÷ (ε × d) × 10⁹] ÷ (Cpr × V_sample ) ÷ T = 3215 × ΔA ÷ Cpr

Parameter Description:

• V_{total reaction}; : Total reaction volume, 2 × 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

• T: Reaction time, 2 min

• Cpr: Sample protein concentration, mg/mL

• W: Sample mass, g

• 500: Cell or bacterial count (5 × 10⁶), converted to units of 1

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 > 1.0, the PK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer or reduce the amount of sample used for extraction, and then re-assay. If the sample ΔA is < 0.005, extend the reaction time to 5 or 10 minutes, or appropriately increase the sample amount, and then re-assay.
Q: Will testing multiple samples simultaneously affect the results?
A: Appropriately extending the time by 3-5 minutes for this assay will not affect the results. When testing multiple samples, it is recommended to use a multi-channel pipette for operation.

Storage and Shipping

Storage

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

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. A1501205A, A1501205B, A1501205D: Store at 2-8℃ long term (12 months). A1501205C: Store at -20℃ long term (12 months). Store in the dark.