Aldolase B Protein, Human (GST)

Materials
Assay buffer: 50 mM Tris-HCl (containing 0.1 mM EDTA), pH 7.4
Substrate solution: 50 mM sodium fructose-1,6-bisphosphate (prepared using assay buffer)
Coenzyme solution: 10 mM β-NADH (prepared fresh with ice-cold ultrapure water)
Coupling enzyme complex: α-Glycerophosphate Dehydrogenase-Triose Phosphate Isomerase (α-GDH-TPI)
Aldolase B Protein, Human (GST) (HY-P74419A)

Procedure
1. Preparation of reaction working solution (per well, total reaction volume 172 μL; prepare an additional 10% volume to compensate for losses)
Component — Volume per well (μL) — Final concentration
Fructose-1,6-bisphosphate (50 mM)——20 μL——50 mM
NADH (10 mM) —— 4 μL —— 0.2 mM
α-GDH-TPI (100 U/mL) —— 2 μL —— 1 U/mL
Assay buffer —— 146 μL—— /
2. Add reaction working solution: Add 172 μL of reaction working solution to each detection well of the 96-well UV plate.
3. Pre-incubation: Place the 96-well UV plate in a pre-warmed microplate reader set to 37°C and incubate for 5-10 minutes to equilibrate the system temperature.
4. Add assay protein: Resuspend protein in sterile water to 200 μg/mL. Prepare 10 μg/mL Aldolase B protein in assay buffer. Quickly add 28 μL of each sample to the wells. Use assay buffer without protein for the control group.
5. Kinetic Monitoring: Immediately return the plate to the microplate reader and initiate absorbance monitoring at 340 nm. Read every 30 seconds for 10 minutes.
6. Calculate Enzyme Activity:
Enzyme activity definition: One enzyme activity unit (U) is defined as the consumption of 1 μmol of NADH per minute.
Enzyme Activity Formula: