Pyruvate Dehydrogenase(PDH) Activity Assay Kit
enzyme labeling method
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| พารามิเตอร์การทดสอบ | ข้อมูลจำเพาะ |
|---|---|
| Performance test | PASS |
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Pyruvate Dehydrogenase (PDH) Activity Assay Kit - Microplate Method
Product Introduction
Pyruvate dehydrogenase (PDH, EC 4.1.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is part of the pyruvate dehydrogenase complex (PDHC), a rate-limiting enzyme complex that catalyzes the oxidative decarboxylation of pyruvate. This reaction generates hydroxyethyl-TPP and links glycolysis with the tricarboxylic acid cycle.
In this assay, PDH catalyzes the dehydrogenation of pyruvate while reducing WST-8 to produce a yellow product, resulting in increased absorbance at 450 nm.
Assay sample used for reference data: corn. Actual readings may vary depending on the testing instrument and assay conditions.
Product Packing List
| Kit Size | Code | Component | Quantity |
|---|---|---|---|
| 100T | 112025.1 | Reagent 1 | 100 mL |
| 100T | 112025.2 | Reagent 2 | 1.5 mL |
| 100T | 112025.3 | Reagent 3 | 20 mL |
| 100T | 112025.4 | Reagent 4 | 17.4 mg |
| 100T | 112025.5 | Reagent 5 | 2.5 mL |
| 100T | 112025.m | Instruction Manual | 1 copy |
Quality Standards and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent 1 | -- | -- |
| Reagent 2 | -- | -- |
| Reagent 3 | -- | -- |
| Reagent 4 | -- | -- |
| Reagent 5 | -- | -- |
Transportation and Storage
| Transport | Transport with ice packs. |
|---|---|
| Storage | Store Reagents III and V at 2-8°C protected from light. Store the other reagents at -20°C. |
| Shelf Life | 180 days. |
Instructions for Use
1. Sample Pretreatment
- Tissue samples: Weigh approximately 0.1 g tissue. Add 1 mL Reagent I and 10 uL Reagent II. Grind and homogenize thoroughly using an ice-bath homogenizer or mortar. Centrifuge at 11000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for testing.
- Cell or bacterial samples: Collect 500 ten thousand bacteria or cells into a centrifuge tube. Centrifuge and discard the supernatant. Add 1 mL Reagent I and 10 uL Reagent II. Disrupt the bacteria or cells by ultrasonic treatment in an ice bath using 200 W power, 3 s ultrasound, 7 s interval, for a total time of 5 min. Centrifuge at 11000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for testing.
- Serum, plasma, and other liquid samples: Test directly. If the solution is turbid, centrifuge and collect the supernatant for measurement.
2. Reagent Preparation
Before use, add 10 mL distilled water to Reagent IV to dissolve it. Store unused portions at -20°C.
3. Measurement Procedure
- Preheat the microplate reader for 30 min or more and set the wavelength to 450 nm.
- Prepare the working solution immediately before use according to the number of samples. Mix Reagent III, Reagent IV, and Reagent V at a ratio of 100 uL : 60 uL : 20 uL.
- Add 20 uL sample and 180 uL working solution to each well of a 96-well plate. Mix well.
- Immediately record the initial absorbance at 450 nm as A1. After 2 min, record the absorbance as A2.
- Calculate ΔA = A2 - A1.
PDH Activity Calculation
The standard curve is y = 6.7928x - 0.0009, R2= 0.9991, where y is ΔA and x is the concentration in umol/mL.
1. Calculation Based on Sample Protein Concentration
Unit definition: One unit of enzyme activity is defined as the amount of enzyme that reduces 1 nmol WST-8 per minute per mg of tissue protein.
PDH (nmol/min/mg prot) = (ΔA + 0.0009) ÷ 6.7928 × Vtotal reaction÷ (Vsample× Cpr) ÷ T × 1000 = 736 × ΔA ÷ Cpr
2. Calculation Based on Sample Fresh Weight
Unit definition: One unit of enzyme activity is defined as the amount of enzyme that reduces 1 nmol WST-8 per minute per g of tissue fresh weight.
PDH (nmol/min/g fresh weight) = (ΔA + 0.0009) ÷ 6.7928 × Vreaction total÷ (W × Vsample÷ Vsample total) ÷ T × 1000 = 743 × ΔA ÷ W
3. Calculation Based on Bacterial or Cell Density
Unit definition: One unit of enzyme activity is defined as the amount of enzyme that reduces 1 nmol WST-8 per minute per 104bacteria or cells.
PDH (nmol/min/104cells) = (ΔA + 0.0009) ÷ 6.7928 × Vreaction total÷ (500 × Vsample÷ Vsample total) ÷ T × 1000 = 1.49 × ΔA
Calculation Parameters
| Vreaction total | Total volume of the reaction system, 0.2 mL |
|---|---|
| Vsample | Volume of sample added, 0.02 mL |
| Vsample total | Volume of extraction solution added, 1.01 mL |
| T | Reaction time, 2 min |
| Cpr | Sample protein concentration, mg/mL |
| W | Sample mass, g |
| 500 | Total number of bacteria or cells, 500 ten thousand |
| 1000 | Conversion factor from umol to nmol |
Precautions
- Before the formal assay, select 2-3 samples with large expected differences for preliminary testing.
- Required instruments and supplies are not included: microplate reader, water bath, benchtop centrifuge, adjustable pipettes, 96-well plate, mortar, ice, and distilled water.
- This kit can assay 96 samples.
Visual Reference
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