Alkaline Phosphatase Assay Kit (Colorimetric) / pNPP assay (ab83369)

Alkaline Phosphatase Assay Kit ab83369 is a quantitative, addition-only alkaline phosphatase (ALP) activity assay. Based on the p-nitrophenyl phosphate (pNPP) assay substrate with 405 nm readout on any standard colorimetric plate reader.

- Cited in over 250 publications.
- Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

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Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (AB83369), expandable thumbnail

Publications

Key facts

Detection method: Colorimetric
Sample types: Plasma, Tissue Extracts, Cell culture media, Serum, Other biological fluids, Cell Lysate
Assay type: Enzyme activity (quantitative)
Reactive species: Mammals
Range: 10 - 250 µU
Assay time: 1h 10m
Sensitivity: > 10 µU

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Target data

See full target information Alkaline phosphatase, tissue-nonspecific isozyme

Function

Alkaline phosphatase that metabolizes various phosphate compounds and plays a key role in skeletal mineralization and adaptive thermogenesis (PubMed:12162492, PubMed:23688511, PubMed:25982064). Has broad substrate specificity and can hydrolyze a considerable variety of compounds: however, only a few substrates, such as diphosphate (inorganic pyrophosphate; PPi), pyridoxal 5'-phosphate (PLP) and N-phosphocreatine are natural substrates (PubMed:12162492, PubMed:2220817). Plays an essential role in skeletal and dental mineralization via its ability to hydrolyze extracellular diphosphate, a potent mineralization inhibitor, to phosphate: it thereby promotes hydroxyapatite crystal formation and increases inorganic phosphate concentration (PubMed:23688511, PubMed:25982064). Acts in a non-redundant manner with PHOSPHO1 in skeletal mineralization: while PHOSPHO1 mediates the initiation of hydroxyapatite crystallization in the matrix vesicles (MVs), ALPL/TNAP catalyzes the spread of hydroxyapatite crystallization in the extracellular matrix (By similarity). Also promotes dephosphorylation of osteopontin (SSP1), an inhibitor of hydroxyapatite crystallization in its phosphorylated state; it is however unclear whether ALPL/TNAP mediates SSP1 dephosphorylation via a direct or indirect manner (By similarity). Catalyzes dephosphorylation of PLP to pyridoxal (PL), the transportable form of vitamin B6, in order to provide a sufficient amount of PLP in the brain, an essential cofactor for enzymes catalyzing the synthesis of diverse neurotransmitters (PubMed:20049532, PubMed:2220817). Additionally, also able to mediate ATP degradation in a stepwise manner to adenosine, thereby regulating the availability of ligands for purinergic receptors (By similarity). Also capable of dephosphorylating microbial products, such as lipopolysaccharides (LPS) as well as other phosphorylated small-molecules, such as poly-inosine:cytosine (poly I:C) (PubMed:28448526). Acts as a key regulator of adaptive thermogenesis as part of the futile creatine cycle: localizes to the mitochondria of thermogenic fat cells and acts by mediating hydrolysis of N-phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation (By similarity). During the futile creatine cycle, creatine and N-phosphocreatine are in a futile cycle, which dissipates the high energy charge of N-phosphocreatine as heat without performing any mechanical or chemical work (By similarity).

Alternative names

AP-TNAP, TNS-ALP, TNSALP, Alkaline phosphatase liver/bone/kidney isozyme, Phosphoamidase, Phosphocreatine phosphatase, ALPL

What's included?

10000 Test

Components

ALP Assay Buffer I

20 x 100 mL

Enzyme Mix II

20 x 1 Each

Stop Solution I

20 x 10 mL

pNPP Substrate

20 x 1 Each

Key facts

Detection method: Colorimetric
Sample types: Plasma, Tissue Extracts, Cell culture media, Serum, Other biological fluids, Cell Lysate
Assay type: Enzyme activity (quantitative)
Reactive species: Mammals
Range: 10 - 250 µU
Assay time: 1h 10m
Assay Platform: Microplate reader
Sensitivity: > 10 µU

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: -20°C
Appropriate long-term storage conditions: -20°C
Storage information: -20°C

Notes

Alkaline Phosphatase Assay Kit (Colorimetric) ab83369 is a highly sensitive, simple, direct and HTS-ready colorimetric assay designed to measure alkaline phosphatase (ALP) activity in serum and other mammalian samples.

How the assay works
The kit is a pNPP assay; it uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λmax= 405 nm) when dephosphorylated by ALP as part of an ALP activity assay. This kit contains 10 substrate tablets providing convenience for multiple usages.

Alkaline phosphatase assay protocol summary

- Add samples and standards to wells
- Add pNPP solution to sample wells (not to standards)
- Add ALP enzyme solution to standard wells (not to samples)
- Incubate for 60 min at room temp
- Add stop solution
- Analyze with microplate reader

Related alkaline phosphatase assay products
For a fluorometric alkaline phosphatase assay kit, we recommend Alkaline Phosphatase Assay Kit (Fluorometric) ab83371. This assay is based on the 4-Methylumbelliferyl phosphate disodium salt (MUP) substrate. Alkaline phosphatase cleaves the phosphate group of this substrate, releasing fluorescent 4-methylumbelliferyl (Ex/Em = 360nm/440nm).

For a luminescent alkaline phosphatase assay, we recommend Alkaline Phosphatase Assay Kit (Luminometric) ab233466. This assay is bsed on the ability of alkaline phosphatase to remove the phosphate group from D-luciferin phosphate, releasing the luciferin to be processed by luciferase.

For alkaline phosphatase staining, we recommend Alkaline Phosphatase Staining Kit Alkaline Phosphatase Staining Kit ab284936. This kit is based on the substrate, 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) which 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.

Getting the best performance from ab83369
Technical Note: Alkaline phosphatase is unlikely to be detectable in healthy urine but may be elevated in certain kidney, liver or bone disorders.

Alkaline phosphatase assay methods
pNPP assays are a class of phosphatase assays using p-nitrophenyl phosphate (pNPP) as a phosphatase substrate. They can be used to measure alkaline phosphatase, neutral phosphatase, and acid phosphatase activity. These different types of phosphatases are active respectively in alkaline, neutral, and acid assay buffers. pNPP assays include Alkaline Phosphatase Assay Kit ab83369 and Acid Phosphatase Assay Kit Acid Phosphatase Assay Kit (Colorimetric) ab83367.

The pNPP phosphatase assay method is by far the most popular method for a colorimetric alkaline phosphatase assay for liquid samples or cell/tissue lysates.

Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K412 Alkaline Phosphatase Activity Colorimetric Assay Kit. K412-500 is the same size as the 500 test size of ab83369.

The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Alkaline phosphatase (ALP) is an enzyme that catalyzes the hydrolysis of phosphate groups from various molecules including nucleotides and proteins. It is a homo-dimeric protein with each monomer having a mass of about 57 kDa. The enzyme is widely expressed across different tissues with high levels present in the liver bone kidney and placenta. ALP is also commonly referred to as ALP or alkaline phosphatase where it exists as various isoenzymes depending on the tissue of expression.

Biological function summary

Alkaline phosphatase is involved in essential processes such as dephosphorylation which impacts cellular communication and energy metabolism. The enzyme participates in bone mineralization by breaking down pyrophosphate an inhibitor of mineralization to facilitate calcium and phosphate deposition. It does not typically function as part of a larger complex but its activity is critical in maintaining normal physiological pH levels in various tissues. The enzyme's activity is often measured using an alkaline phosphatase assay or ALP assay valuable for assessing different condition states within the body.

Pathways

Alkaline phosphatase plays roles in bone formation and liver function pathways. It interacts with other proteins like collagen and osteocalcin in the bone mineralization pathway which is vital for healthy bone development and maintenance. Additionally in liver function ALP is linked to other enzymes like gamma-glutamyltransferase (GGT) which together help detoxify hematopoietic and other bodily functions. Alkaline phosphatase activity can influence these pathways significantly impacting health status and treatment outcomes.

Associated diseases and disorders

Abnormal alkaline phosphatase levels often link to conditions such as Paget's disease of bone and hepatobiliary dysfunction. In Paget's disease elevated ALP indicates increased bone turnover often with increased osteocalcin activity. Meanwhile in liver disease altered ALP levels may connect with gamma-glutamyltransferase during diagnostic analyses to assess liver dysfunction or biliary obstruction. Regular monitoring of ALP activity is important for diagnosing these conditions and evaluating treatment responses.

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9 product images

Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
ALP measured in cell culture supernatants showing activity (U) per L of tested sample. Samples were diluted 1-16 fold.
Image courtesy of Hermenean A et al. Sci Rep. 2017; 7: 16641. doi: 10.1038/s41598-017-16599-5
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
ALP activity in cells undergoing osteogenic differentiation.
Hermenean A et al. (2017) measured alkaline phosphatase activity in cells cultured in CHT/GO (3D chitosan scaffolds enriched with graphene oxide) biomaterials for up to 28 days using ab83369.
Image courtesy of Alesutan I et al. Sci Rep. 2017; 7: 2059. doi: 10.1038/s41598-017-01882-2. Reproduced under the creative commons license http://creativecommons.org/licenses/by/4.0/.
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Effects of mineralocorticoid receptor blockade on phosphate-induced vascular calcification in vitro.
Alesutan I et al. (2017) measured alkaline phosphatase activity in vitro in primary human aortic smooth muscle cells using ab83369. Cells were treated with phosphate (pi) or without (Ctr, and with or without additional treatment of spironolactone (Spr) or 10 μM eplerenone (Epl).
Image from Lee YM. et al., Biomed Res Int. 2019 Feb 19;2019:5073085. Fig 6b doi: 10.1155/2019/5073085. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Alkaline phosphatase activity measured in the study of osteoporosis.
Lee YM et al. (2019) used alkaline phosphatase assay kit ab83369 to measure the serum levels of alkaline phosphatase as a marker of bone turnover. Rat groups include sham-operated (Sham), OVX-control (OVX), E2-treated OVX groups (E2), and LABE 500 mg/kg treated OVX groups (LABE) (N=10/group).
Serum ALP levels in the OVX group were significantly increased compared to those in Sham group, and oral administration of LABE (500 mg/kg) significantly reduced the serum ALP levels as an osteoporosis-related serum marker.
The serum levels of alkaline phosphatase (ALP) were measured using assay kits (ab83369, Alkaline Phosphatase Assay Kit; Abcam) according to the manufacturer's instructions. Briefly, 80 μL of sample dilutions, 50 μL of 5 mM p-nitrophenyl phosphate (pNPP) solution, and 10 μL of ALP enzyme solution were added sequentially to the reaction wells and the plate was incubated at 25°C for 60 min protected from light. The reaction was halted by adding 20 μL stop solution, and then the absorbance was measured at 405 nm. ALP activity was calculated using a standard curve.
Image from Nepelska M et al., PLoS One 7(12), fig 8c. Doi: 10.1371/journal.pone.0052869. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Nepelska M et al. (2012) assayed alkaline phosphatase activity in Caco-2 cells after 48 hours treatment with PMA and Butyrate using Alkaline Phosphatase assay kit (ab83369).
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Standard curve: mean of duplicates (+/- SD) with background reads subtracted
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
ALP measured in biological fluids showing activity (U) per L of tested sample. Samples were diluted 4-64 fold.
Functional Studies - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Measurement of ALP activity using ab83369. a. ALP activity in fresh medium (80 μL, without culturing), 3 day old HeLa cell culture medium (80 μL) and human serum (80 μL, 1/10 dilution). b. ALP activity in HeLa cells. 5 x 10⁴ HeLa cells were homogenized in 1 mL of Assay Buffer, diluted 1/10 in Assay Buffer, and 80 μL used for the measurement.
Biochemical assay - Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)
Diagram showing the principles of the Alkaline phosphatase assay method.