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: Other species, Mammals
  • 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.

    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
    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:
  • Xie L  et al. Potential Biomarkers for Primary Open-Angle Glaucoma Identified by Long Noncoding RNA Profiling in the Aqueous Humor. Am J Pathol 189:739-752 (2019). Read more (PubMed: 30677397) »
  • Morsi NM  et al. Risedronate-Loaded Macroporous Gel Foam Enriched with Nanohydroxyapatite: Preparation, Characterization, and Osteogenic Activity Evaluation Using Saos-2 Cells. AAPS PharmSciTech 20:104 (2019). Read more (PubMed: 30737611) »
See all 114 Publications for this product

Customer reviews and Q&As

21-30 of 32 Abreviews or Q&A

Answer

The Tris buffer should be fine, but the Triton-X will not be good to use. Ideally you would extract the cells off of the scaffold without lysing them, then resuspend them in the assay buffer and proceed with the protocol.

I would recommend trypsinizing the cells off the scaffold, then splitting the cells if some are needed for other assays, and resuspending a portion of the cells in the assay buffer.

Read More

Answer

I can confirm that the ALP Enzyme in this kit is from calf intestine.

Read More

Answer

1) The cells lysed in the ALP assay buffer cannot ideally be used formeasuring DNA and extracting proteins.

2) I am not certain that the assay buffer will digest the scaffolding. Cells may need to be extracted out of scaffold and then processed with the assay buffer.

3 and 4) Use 100 µl of assay buffer with each 10^5 cells with a dounce homogenizer.

Read More

Answer

This kit has been tested and characterized with different types of samples such as urine, serum, plasma, tissue extracts, cell lysate, cell culture media. However, we have never used IP pull- down samples. As for all the enzymes, pH is very crucial. The optimal pH for the ALP activity is around at 8- 8.5 depending on the species (E.coli, human, bovine etc). Once the pH has changed from alkalic to acidic range, it may well be that the enzyme activity has been changed irreversibly.

Read More

Answer

In this case,washed cells (1×10^5) can be homogenized in the Assay Buffer and centrifuged to remove insoluble material at 13,000g for 3 minutes. The supernatant from this can be aliquoted and stored at -80°C for later analysis. The aliquoting is important because repeated freeze thaw will degrade the enzyme.

Read More

Question
Answer

The buffer contained in the kit does not contain detergents, so mechanical agitation is required to complete the process of lysing the cells by passing through a syringe tip or other utensil such as a homogenizer. The scrapper is not recommended in this case.

Read More

Answer

The procedure is as follows:

1. Wash tissue: 10 mg or cells 1X106 cell in ice cold PBS.
2. Spin at 1000 g for 5 mins at 4C.
3. Resuspend and homogenize in ˜2-3 volumes of the assay buffer (use Dounce homogenizer, if needed to ensure cell lysis) on ice.
4. Spin at 13,000 g for 10 min at 4C.
5. Collect the supernatant and test different volumes for the assay.

Read More

Answer

The Assay buffer and Stop buffer could be stored at -20C. Please make sure they are at room temperature when using.
The assay buffer and Stop solution will be stable for 1 year.

Read More

Question
Answer

The kit works by measuring the Alkaline Phosphatase activity of a sample through the monitoring of the dephosphorylation of the p-nitrophenyl phosphate (pNPP) phosphatase substrate, producing a yellow colour (Lambda max= 405 nm). As long as your sample contains Alkaline Phosphatase then the kit should work with mouse samples.

Read More

Answer

Tissue samples can also be used with this kit and can be treated in a similar manner to cell samples. 
For tissue samples:
- Start with 10-100 mg of the tissue (rinse well in PBS),
- add 500-1000 μl (or ~3-4 volumes) of the assay buffer on ice,
- homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope.
- Spin down the sample and collect the supernatant.
- Load the supernatant unto a 10kda spin column for deproteinzation (if indicated in the data sheet; not required for enzyme assays).
- Use the eluate for your subsequent assays.
- Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve. 

Read More

21-30 of 32 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up