Description
Phenylalanine Ammonia Lyase (PAL) Activity Assay Kit
This kit can be used to measure phenylalanine ammonia lyase (PAL) activity in plant tissue samples.
| Product Name: | Phenylalanine Ammonia Lyase (PAL) Activity Assay Kit |
| SKU: | MAES0426 |
| Size: | 100 Assays |
| Detection Wavelength: | 290 nm |
| Sensitivity: | 0.78 U/g tissue |
| Sample Type: | Plant tissue samples |
| Assay Time: | 30 minutes |
| Storage: | 2-8°C, 12 months |
Phenylalanine ammonia lyase (PAL) catalyzes L-phenylalanine to produce trans-cinnamic acid and ammonia. Trans-cinnamic acid has a maximum absorption peak at 290 nm. PAL activity can be calculated by measuring the increase in optical density at 290 nm.
| Item | Component | Size 1 (50 Assays) | Size 2 (100 Assays) | Storage |
| Reagent 1 | Extracting Solution | 30 mL × 1 vial | 60 mL × 1 vial | 2-8°C, 12 months |
| Reagent 2 | Buffer Solution | 50 mL × 1 vial | 50 mL × 2 vials | 2-8°C, 12 months |
| Reagent 3 | Substrate Powder | × 2 vials | × 4 vials | 2-8°C, 12 months |
| Reagent 4 | Stop Solution | 3 mL × 1 vial | 6 mL × 1 vial | 2-8°C, 12 months |
1. Sample preparation:
- Harvest the amount of tissue needed for each assay (initial recommendation 20 mg).
- Wash tissue in cold PBS (0.01 M, pH 7.4).
- Homogenize 20 mg tissue in 180 μL extracting solution with a dounce homogenizer at 4°C.
- Centrifuge at 10,000×g for 10 minutes to remove insoluble material. Collect supernatant and keep it on ice for detection.
- If there is any floating matter, take the supernatant and centrifuge it again until the supernatant is completely clarified.
2. Sample dilution: The recommended dilution factor for different samples is as follows (for reference only):
- 10% Epipremnum aureum tissue homogenate: 1
- 10% Carrot tissue homogenate: 1
- 10% Green pepper tissue homogenate: 1
- 10% Corn grain tissue homogenate: 1
Note: The diluent is extracting solution. For dilution of other sample types, please perform a pretest to confirm the dilution factor.
- Control tube: Add 800 μL of buffer solution and 200 μL of substrate working solution into 1.5 mL EP tubes. Sample tube: Add 20 μL of sample, 780 μL of buffer solution, and 200 μL of substrate working solution into 1.5 mL EP tubes.
- Mix thoroughly with the vortex mixer for 3 seconds, then incubate at 37°C for 30 minutes.
- Add 40 μL of stop solution to each tube.
- Mix thoroughly with the vortex mixer for 3 seconds, stand for 5 minutes, then set to zero with double distilled water and measure the OD value of each tube with a 1 cm optical path cuvette at 290 nm.
Definition: One unit of enzyme activity is defined as the amount of enzyme that causes a 0.1 OD change per minute per gram of tissue in 1 mL of reaction system at 37°C.
Formula: PAL activity (U/g tissue) = ΔA_290 × V2 ÷ 0.1* ÷ t ÷ (m ÷ V3 × V1) × f
Note:
- ΔA_290: ODsample - ODcontrol
- m: weight of sample (recommended 0.05 g)
- V1: volume of sample added to the reaction (0.02 mL)
- V2: total volume of the reaction system (1.04 mL)
- V3: volume of extracting solution added (if m=0.05 g, then V3=0.45 mL)
- t: enzymatic reaction time (30 min)
- *: the absorbance value decreased by 0.1
- f: dilution factor of sample before test
