Description
Key facts
Detection method:Fluorescent,
Sample types:Saliva, Urine (UTI), Plasma, Tissue Extracts, Cell culture media, Serum, Other biological fluids, Cell Lysate,
Assay type:Enzyme activity (quantitative),
Assay time:1h,
Assay Platform:Microplate reader
Product details:
Alkaline Phosphatase Assay Kit (Fluorometric) (ab83371) is a highly sensitive kit to detect alkaline phosphatase (ALP) activity in serum and other biological samples. In the alkaline phosphatase assay protocol, ALP cleaves the phosphate group of the non-fluorescent 4-Methylumbelliferyl phosphate disodium salt (MUP) substrate, resulting in an increased fluorescent signal (Ex/Em = 360nm/440nm) when the substrate is dephosphorylated.
The kit is an ultra sensitive, simple, direct and HTS-ready assay designed to measure ALP activity with a detection sensitivity ~1 μU, more sensitive than colorimetric assays.
Alkaline phosphatase assay protocol summary:
- add samples and standards to wells
- add reaction mix to wells
- add ALP enzyme solution to standard wells (not to samples)
- incubate for 30 min at room temp
- add stop solution
- analyze with microplate reader
Technical Note: ALP is unlikely to be detectable in healthy urine but may be elevated in the presence of infection.
Related alkaline phosphatase assay products
For a colorimetric alkaline phosphatase assay kit, we recommend
ab83369
. which is based on the p-nitrophenyl phosphate (pNPP) assay substrate. pNPP turns yellow (405 nm) when dephosphorylated by ALP as part of an ALP activity assay.
For a luminescent alkaline phosphatase assay, we recommend
ab233466
, which is based on the ability of alkaline phosphatase to remove the phosphate group from D-luciferin phosphate. This releases the luciferin to be processed by luciferase.
For alkaline phosphatase staining, we recommend Alkaline Phosphatase Staining Kit
ab284936
, which is based on the substrate, 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) substrate. BCIP is processed by alkaline phosphatase to form a blue colored intermediate. Nitro Blue Tetrazolium (NBT) then oxidizes the intermediate to form an insoluble, dark blue dimer, which can be viewed by microscopy.
This product is manufactured by BioVision, an Abcam company and was previously called K422 Alkaline Phosphatase Activity Fluorometric Assay Kit. K422-500 is the same size as the 500 test size of ab83371.
Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. The change in alkaline phosphatase level and activity is associated with many diseases in the liver and bones.
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Alkaline phosphatase also known as ALP is an enzyme with significant biological roles. It mechanically functions by catalyzing the hydrolysis of phosphate groups from various molecules including nucleotides proteins and alkaloids under alkaline conditions. ALP is a metalloenzyme possessing zinc and magnesium ions at its active site to aid in the reaction. This enzyme has a molecular mass of approximately 86 kDa and exists in dimeric form. ALP is expressed in many tissues with high levels in liver bone kidney and the placenta.
Biological function summary
ALP plays an important role in maintaining mineral balance and bone metabolism by breaking down pyrophosphate an inhibitor of mineralization. It facilitates the regulation of phosphate metabolism by contributing to phosphorus and calcium homeostasis. ALP is not part of a larger complex but interacts with calcium and phosphate ions to aid in the formation and resorption of bone mineral tissue. It serves as a marker in bone growth and liver function due to its prominent activity in those tissues visible in assays like the ALP staining method used to gauge enzyme presence and action.
Pathways
Alkaline phosphatase activity plays an essential role in the metabolic pathways related to bone mineralization and liver function. In bone tissue the enzyme participates in the calcification process important for bone strength and development by regulating inorganic phosphate availability. The liver expression of ALP is involved in bile excretion pathways where it dephosphorylates bile acids aiding in their conversion and recycling. It has a significant relationship with proteins such as osteocalcin in the bone mineralization pathway and bile acid transporters in the liver.
Alkaline phosphatase levels serve as diagnostic indicators for bone and liver diseases. High levels of ALP are often associated with bone disorders like Paget's disease and liver conditions like cholestasis. In these disorders ALP activity rises due to increased enzyme synthesis by osteoblasts in bone diseases or impaired bile flow in liver diseases. Furthermore the liver enzyme gamma-glutamyl transferase (GGT) is often measured alongside ALP to differentiate the cause of elevated ALP levels particularly to assess liver pathology.
