Fructose-1, 6-Bisphosphate (FBP) is a primary intermediate in cellular carbohydrate metabolism serving as a vital signaling metabolite for cell adhesion and membrane protrusion. It serves as a central energy regulator in glycolysis and gluconeogenesis. Deficiency of FBP is usually caused by upstream enzyme deregulation, notably phosphofructokinase-1 deficiency, which results in metabolic muscle crises like rhabdomyolysis. Accumulation of FBP is the result of downstream aldolase deficiencies leading to hypoglycemia, cell toxicity, and acute liver damage. Lack of FBP worsens ischemia tissue damage and loss of anti-inflammatory signals due to the body not being able to generate adenosines and suppress inflammatory cytokines. Measuring FBP levels is critical as a proxy for cellular energy production and metabolic health.

Fructose-1, 6-Bisphosphate Assay Kit provides a simple, reproducible, and sensitive tool of measuring FBP concentration in cell lysates and other biological liquid samples. This assay is based on a coupled enzyme reaction. Fructose-1, 6-bisphosphate is catalyzed by aldolase to a colored product which can be quantified using a spectrophotometer or a microtiter plate reader at 450 nm. The optical density measured at 450 nm is directly proportional to the concentration of FBP present in the sample.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• Orbital shaker capable of 37°C
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted FBP Standards or samples to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Seal the plate with a plate sealer or plastic wrap to reduce evaporation. Incubate the plate for 40 minutes at 37°C with mixing in the dark.
5. Measure the absorbance at 450 nm using a plate reader.