Alkaline phosphatase (ALP) is an enzyme that catalyzes the hydrolysis of phosphate esters in an alkaline environment, leading to the release of inorganic phosphate. This inorganic phosphate is crucial for various biological processes, including bone mineralization, glycolysis, and oxidative phosphorylation. Alterations in serum ALP levels are indicative of specific diseases and have been utilized as a biomarker for liver and bone disease, such as Paget’s disease and Hyperphosphatasemia. Additionally, a significant increase in serum ALP levels has been associated with conditions like malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis.

RayBio® Alkaline Phosphatase (ALP) Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring Alkaline Phosphatase (ALP) Activity in plasma, serum, cell lysates, urine and other biological liquid samples. The assay involves monitoring the dephosphorylation of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by ALP. First, ALP dephosphorylates pNPP, leading to the formation of p-nitrophenol. Then the phenolic hydroxyl group undergoes deprotonation under alkaline conditions, resulting in the generation of p-nitrophenolate. This reaction yields a yellow-colored product that can be quantified at 405nm. The rate of the reaction is directly proportional to the enzyme activity.

• Microplate reader capable of measuring absorbance at 405 nm
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C.
• 50 ml conical tubes
• Distilled or deionized water

Each Alkaline Phosphatase Standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add solutions to each well: (a) For Blank well(s) (Standard 0 U/L): add 30 μL ALP Assay Buffer. (b) For Samples and standards: add 10 μL ALP Assay Buffer and 20 μL sample/standard.
2. Initiate the reaction by adding 20 μL reconstituted ALP Substrate (See “Reagent Preparation”) to each well.
3. Cover with the plate cover. Carefully shake the plate for a few seconds to mix.
4. Incubate the plate for 20 minutes at 37oC in the dark.
5. Pipette 100 μL of 1X ALP Stop Solution to each well to stop the reactions.
6. Measure the absorbance at 405nm immediately using a plate reader.