Jacobs:Protocol Alkaline Phosphatase Assay

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Alkaline Phosphatase Assay


  • -20 freezer
    • RIPA lysis kit
      • PMSF solution
      • Protease inhibitor cocktail
    • Alkaline Phosphatase Fluorometric Assay Kit (Sigma Aldrich - APF; Abcam - ab83371)
      • Control enzyme
      • Substrate
  • Fridge
    • Buffers from APF kit
  • Miscellaneous
    • 96 well plate
    • 1.5 ml microcentrifuge tubes
    • Pipette, tips


  1. Buffer
    • 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample
    • Prepare enough of the working buffer for the standards and unknowns
  2. Standards
    • Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase.
  3. Samples
    • Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul.


  1. Prepare the wells by adding 20 ul of each standard or sample in triplicate.
  2. Add 180 ul of the working buffer to each well.
  3. Add 1 ul of the substrate to each well. Do this quickly as the substrate will immediately be converted.
  4. Measure fluorescence in the plate reader at 330 nm excitation and 440 nm. Generate a standard curve of FSU vs. alkaline phosphatase in ug/ml.


  • 11/2/10 - Triton-X works much better than RIPA. RIPA may affect the protein-protein interaction.
  • 11/15/10 - Colorimetric assay version of this protocol using substrate buffer and p-nitrophenol did not work well with MC3T3s, MLOY4s or primary osteoblasts. It is likely because it is not as sensitive as the fluorimetric assay.