Overview

  • Product name
    Alkaline Phosphatase Assay Kit (Colorimetric)
    See all Alkaline phosphatase kits
  • Detection method
    Colorimetric
  • Sample type
    Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate, Cell culture media
  • Assay type
    Enzyme activity (quantitative)
  • Sensitivity
    > 10 µU
  • Range
    10 µU - 250 µU
  • Assay time
    1h 10m
  • Product overview

    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.


    The kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λmax= 405 nm) when dephosphorylated by ALP.


    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

  • Notes

    This kit contains 10 substrate tablets providing convenience for multiple usages.

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components Identifier 500 tests
    ALP Assay buffer NM 1 x 100ml
    ALP Enzyme (Lyophilised) Green 1 vial
    pNPP Substrate (10 tablets) Red 1 vial
    Stop Solution WM 1 x 10ml
  • Research areas
  • Relevance
    Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. Changes in alkaline phosphatase level and activity are associated with various disease states in the liver and bone.
  • Cellular localization
    Cell membrane; Lipid-anchor, GPI-anchor.
  • Alternative names
    • Alkaline phosphatase intestinal
    • Alkaline phosphatase liver/bone/kidney
    • Alkaline phosphatase liver/bone/kidney isozyme
    • Alkaline phosphatase tissue nonspecific isozyme
    • Alkaline phosphomonoesterase
    • ALPI
    • ALPL
    • AP TNAP
    • Glycerophosphatase
    • HOPS
    • IAP
    • Intestinal alkaline phosphatase
    • Kasahara isozyme
    • Liver/bone/kidney type alkaline phosphatase
    • Tissue nonspecific ALP
    • TNAP
    • TNSALP
    see all

Associated products

Images

  • Alkaline phosphatase was measured in Caco-2 cells after 48 hours treatment with PMA and Butyrate using Alkaline Phosphatase assay kit (ab83369).

  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • ALP measured in cell culture supernatants showing activity (U) per L of tested sample. Samples were diluted 1-16 fold.

  • ALP measured in biological fluids showing activity (U) per L of tested sample. Samples were diluted 4-64 fold.

  • 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.

Protocols

References

This product has been referenced in:
  • Hosseini S  et al. Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells. Colloids Surf B Biointerfaces 173:662-671 (2019). Read more (PubMed: 30368214) »
  • Jahangir S  et al. 3D-porous ß-tricalcium phosphate-alginate-gelatin scaffold with DMOG delivery promotes angiogenesis and bone formation in rat calvarial defects. J Mater Sci Mater Med 30:1 (2018). Read more (PubMed: 30564959) »
See all 81 Publications for this product

Customer reviews and Q&As

1-10 of 32 Abreviews or Q&A

Answer

I confirm this kit can be used with both protein assay kits (BCA and Bradford).

Read More

Answer


To prepare the samples they should not necessarily require sonication, so I can recommend to follow the protocol.
If there are difficulties lysing the sample, then sonication can be tried. But it is not usually required.

Read More

Answer

We have not tested the kit with bone samples, but there is no reason for the kit to not work with bone samples. Grinding and homogenization would be critical. The following two references used ab83369 to detect alkaline phosphatase produced in bone tissue engineering applications, so it seems the kit was detecting alkaline phosphatase produced by osteogenic progenitor cells:



http://www.ncbi.nlm.nih.gov/pubmed/23804651?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/23022346?dopt=Abstract



If you need bone alkaline phosophatase antibodies we carry the two antibodies below, which are both tested and guaranteed to work in ELISA in the species listed on their datasheets for 12 months after your purchase:



https://www.abcam.com/products?keywords=bone+alkaline+phosphatase

Read More

Answer

Unfortunately, the full composition of the buffer is proprietary however, I can tell you that it does contain Brij35 as a detergent to lyse cells but also zinc chloride.

Read More

Answer

To measure intracellular ALP, washed cells (˜100,000 cells) can be homogenized, preferably using a dounce homogenizer in the Assay Buffer (2-3 times the volume of the cells). Then centrifuge to remove insoluble material at 13,000g for 3 minutes at 4 deg C. ITs recommended to use different volumes of the supernatant to determine optimum sample volume per well for each sample. Bring the total volume to 80 ul with Assay Buffer. The optimized volume should give numbers within the linear range of the standard curve.

Read More

Answer

10ul of enzyme is used for each well for the standards only. There is 1ml of reconstituted enzyme which is equal to 100 standard wells. Each std has 6 datapoints. With the enzyme mix the standard curve can be run 16 times.
The samples do not need any enzyme since this assay is for testing ALP enzyme activity in the sample.

Read More

Answer

That note refers to cell culture medium samples, and the control would be a sample of medium that has not been exposed to cells. If your samples are cell lysates, you do not need this control. Your negative control will simply be the Assay Buffer included with the kit, which is the zero standard.

Read More

Answer

Based on the standard curve equation, y=mx+b, you have to calculate x. This number signifies umoles of pNP for 1 OD. This number is A.

Read More

Answer

The experimental samples are incubated with pNPP for only 60 minutes before adding stop solution. That is also the case for the incubation of the pNPP standards with the ALP enzyme: 60 minutes of incubation, then stop solution.

Read More

Question
Answer



Yes, and it can be found in our catalogue as ALP Enzyme (ab134678).

Read More

1-10 of 32 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up