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Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)

Submit a review Q&A (32)References (273)
ALP activity in cells undergoing osteogenic differentiation
  • Effects of mineralocorticoid receptor blockade on phosphate-induced vascular calcification in vitro
  • Alkaline phosphatase activity measured in the study of osteoporosis
  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)

Key features and details

  • Assay type: Enzyme activity (quantitative)
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 1 hr 10 min
  • Sample type: Cell culture media, Cell Lysate, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine
  • Sensitivity: 10 µU

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

  • Species reactivity

    Reacts with: Mammals, Other species

  • 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 is a pNPP assay; it 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 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.

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

    pNPP Assays

    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 ab83367.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 500 tests 10000 tests
    ALP Assay Buffer I NM 1 x 100ml 20 x 100ml
    Enzyme Mix II White 1 vial 20 vials
    pNPP Substrate Red 1 vial 20 vials
    Stop Solution I WM 1 x 10ml 20 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

  • ALP activity in cells undergoing osteogenic differentiation
    ALP activity in cells undergoing osteogenic differentiationImage courtesy of Hermenean A et al. Sci Rep. 2017; 7: 16641. doi: 10.1038/s41598-017-16599-5

    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.

  • Effects of mineralocorticoid receptor blockade on phosphate-induced vascular calcification in vitro
    Effects of mineralocorticoid receptor blockade on phosphate-induced vascular calcification in vitroImage 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/.

    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).

  • Alkaline phosphatase activity measured in the study of osteoporosis
    Alkaline phosphatase activity measured in the study of osteoporosisImage 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/

    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. 

  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
    Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)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/

    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 Colorimetric Assay Kit (ab83369)
    Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)

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

  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
    Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)

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

  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
    Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)

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

  • Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (ab83369)
    Functional Studies - Alkaline Phosphatase Colorimetric Assay Kit (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.

References (273)

Publishing research using ab83369? Please let us know so that we can cite the reference in this datasheet.

ab83369 has been referenced in 273 publications.

  • Haseli M  et al. Crosslinked Layered Surfaces of Heparin and Poly(L-Lysine) Enhance Mesenchymal Stromal Cell Behavior in the Presence of Soluble Interferon Gamma. Cells Tissues Organs 212:8-20 (2023). PubMed: 34937023
  • Jia CQ  et al. A biomimetic gradient porous cage with a micro-structure for enhancing mechanical properties and accelerating osseointegration in spinal fusion. Bioact Mater 23:234-246 (2023). PubMed: 36439084
  • Shi H  et al. MicroRNAs in Serum Exosomes as Circulating Biomarkers for Postmenopausal Osteoporosis. Front Endocrinol (Lausanne) 13:819056 (2022). PubMed: 35360081
  • Kotlarz M  et al. Bioprinting of Cell-Laden Hydrogels onto Titanium Alloy Surfaces to Produce a Bioactive Interface. Macromol Biosci 22:e2200071 (2022). PubMed: 35365963
  • Fakhr Y  et al. Tumor Necrosis Factor-α Induces a Preeclamptic-like Phenotype in Placental Villi via Sphingosine Kinase 1 Activation. Int J Mol Sci 23:N/A (2022). PubMed: 35409108
View all Publications for this product

Customer reviews and Q&As

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1-10 of 32 Abreviews or Q&A

Question

Is the cell lysis buffer of the ab83369 kit compatible with common protein assays such as bradford of BCA?

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Answer

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

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Question


i have a question about ALP kit (ab83369). For cells preparation before test, cells don't need to be sonicated? Cells suspension must be just washed and centrifuged at top speed at 4oC. Exactly?

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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.

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Question

I would appreciate if you can let me know whether you elisa kit Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369),
can identify bone alkaline phosphatase.

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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

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Question

I need to know more details about the kit buffer composition. I need to know if in the buffer there's the addition of Zn ions

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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.

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Question

My sample for use with this assay will be a cell extract. However the protocol does not provide details on how to create this extract. Specifically, how should washed cells be homogenized in Assay Buffer?

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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.

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Question

In the manual, it is written that the ALP enzyme should reconstituted with 1 mL of buffer and in each sample we should add 10 microliter of enzyme. That means we can analyze 100 sample (not 500) with this kit because there is only one vial of enzyme inside the kit. Could you please explain in more detail how it is possible to get 500 tests out of this kit?

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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.

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Question

We are using ab83369 to determine ALP in cell samples. In the assay protocol it is recommended to use a sample background control to may be used for background subtraction. What type of sample would be appropriate for this? Should this be a cellular sample of some sort, or are you referring to a colorless sample, such as water that is used solely for color subtraction?

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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.

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Question

I am using kit ab83369 but the data analysis section is a bit confusing. How is the 'A' variable determined (described as 'amount of pNP generated by samples (in umol)'?

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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.

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Question

In the protocol, the samples are prepared first and then incubated with pNPP for 60 minutes. Then the standards are prepared and incubated with ALP enzyme for 60 minutes. Does that mean the samples are incubating for another 60 minutes?

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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.

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Question

Is there a way to purchase/obtain this enzyme separately?

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Answer



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

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1-10 of 32 Abreviews or Q&A

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