PLA2G1B Protein, Mouse (HEK293, His)

Materials
Assay buffer: 50 mM HEPES, 1 mg/mL BSA, pH 7.0
Substrate buffer: 50 mM HEPES, 20 mM CaCl?, 1 mg/mL BSA, pH 7.0
PLA2G1B Protein, Mouse (HEK293, His) (HY-P71212)
Trypsin
Substrate: 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (16:0-Pyr PC). Dilute to 2 mM in DMSO first, then dilute to a final stock concentration of 400 μM in ethanol
Standard: 1-Pyrenedecanoic Acid

Procedure
1. Preparation of standard curve: Dilute the standard with assay buffer to concentrations of 0, 0.1, 1, 2.5, 5, 10, 25, 50, and 100 μM. Add 100 μL of each dilution to a 96-well black microplate, and read the fluorescence intensity in endpoint mode with excitation and emission wavelengths of 345 nm and 395 nm (top read).
2. Thaw the frozen substrate at 37°C for 5 minutes and mix well.
3. Dilute Mouse PLA2G1B protein to 100 μg/mL in assay buffer.
4. Dilute trypsin to 0.5 μg/mL in assay buffer.
5. Mixture preparation: Mix equal volumes of diluted trypsin and Mouse PLA2G1B protein, and incubate at room temperature for 30 minutes.
6. Dilute the substrate to 100 μM in substrate buffer.
Experimental group: 50 μL mixture + 50 μL substrate.
Blank group: 50 μL assay buffer + 50 μL substrate.
7. Read in kinetic mode for 5 minutes with excitation and emission wavelengths of 345 nm and 395 nm (top read) respectively.
8. Calculate the specific activity: