Phenylalanine Ammonia Lyase (PAL) Activity Assay Kit (MAES0426)

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.

1. Sample preparation:

1. Harvest the amount of tissue needed for each assay (initial recommendation 20 mg).
2. Wash tissue in cold PBS (0.01 M, pH 7.4).
3. Homogenize 20 mg tissue in 180 μL extracting solution with a dounce homogenizer at 4°C.
4. Centrifuge at 10,000×g for 10 minutes to remove insoluble material. Collect supernatant and keep it on ice for detection.
5. 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.

1. 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.
2. Mix thoroughly with the vortex mixer for 3 seconds, then incubate at 37°C for 30 minutes.
3. Add 40 μL of stop solution to each tube.
4. 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