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Alkaline Phosphatase Activity Colorimetric Assay Kit

based on 9 citations in multiple journalsAlkaline Phosphatase Activity Colorimetric Assay Kit94.2 4
Highly Sensitive Assay, HTS
Catalog #: K412
$415.00

Product Details

Cat # +Size K412-500
Size 500 assays
Detection Method Absorbance (405 nm)
Species Reactivity Mammalian
Applications The Kit can detect 10-250 µU ALP in samples.
Features & Benefits • Simple procedure; takes ~ 3 hours
• Fast and convenient
• The assay is sensitive and stable
• The TUNEL-based assay kit provides complete components including positive and negative control cells for convenient detection of DNA fragmentation in cultured cells and tissue sections.
Kit Components • ALP Assay Buffer
• pNPP (10 TAB)
• ALP Enzyme
• Stop Solution
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

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. BioVision’s Alkaline Phosphatase Assay Kit is a highly sensitive, simple, direct and HTS-ready colorimetric assay designed to measure ALP activity in serum and biological samples. It contains 10 substrate tablets providing convenience for multiple usages. The kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λmax= 405 nm) when dephosphorylated by ALP. The Kit can detect 10-250 µU ALP in samples.


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We would like to used this kit with protein samples isolated that contain protease inhibitor vs samples that do not contain protease inhibitor. Are there any other components that she should be aware of that could affect the performance of this kit when one uses protease inhibitors? Which protease inhibitor is recommended for use in this Alkaline phosphastase assay kit?
The only chemicals you need to be wary of are EDTA, oxalate, fluoride, and citrate. There is no recommended protease inhibitor for this assay kit. You can use our K271-500 cocktail.
Once the pnPP solution has been prepared, can I freeze the pnPP solution (e.g. at -20 degrees Celcius) so that I can use it later? If so, what is the maximum time I can store it at -20 degrees Celcius?
The pNPP solution unfortunately should be used up within 12 hrs of making. Once you collect all samples, you can make the fresh pNPP solution and use it right away.
We wish to measure samples at different time points. So, during the sample preparation, once I have added ALP assay buffer to my cell samples, can I then freeze these samples to -20 degrees Celcius?
Samples homogenized in the assay buffer can be frozen in aliquots at -80 degrees Celcius until analysis
Can the incubation steps for pNPP and the ALP enzyme be combined into a single one hour incubation step? Or is it necessary to let the two incubations take place separately.
Yes, you can do the incubation simultaneously.
I was thinking to keep the volume (and number) of cells stable and change the dose concentration of the drug I am testing with this assay. Why do you recommend different cell volumes?
We are not taking different volume of cells, but different vol of the cell homogenate. So in a nut shell, you plate equal number of cells in each well, treat them with the drugs, then trypsinize out the cells, get the cell pellet, wash with ice cold PBS, homogenize the resultant pellet in the assay buffer, take the supernatant from that and use different volumes of this supernatant for the subsequent assay.
How do we normalize our final readings?
If you are beginning with variable number of cells, you can normalize against the total cell number or protein quantity used.
We are looking for a kit to detect secreted alakaline phosphatase reporter gene in serum samples. Can we use this kit?
If you want to assay for the gene, you need to do DNA isolation from the serum and use a PCR based reaction to detect the specific gene. This assay is to detect the enzyme activity. A DNA isolation kit from serum samples and some SEAL specific primers would be ideal for you.
When the enzyme is resuspended and stored at 4 degrees Celcius what is the reduction of viability of this when used at time points after the two months recommended, for example three or four months later?
We have not checked the enzyme stability statistics at 4 degrees Celcius for longer than 2 months. There is a fair chance that it will be stable for slightly longer, but I do not know exactly how long and then after that time how much of the stability/efficiency would be lost.
I would like to ask you what is the origin of the standard ALP enzyme that comes with the kit (human, bovine, recombinant)?.
The enzyme comes from calf intestine.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20 degrees Celcius. Please refer to the datasheet for storage information and shelf life of each of the components.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Mohamed Aboul Ezz et al., (2017) The effect of cholesterol loaded cyclodextrins on post-thawing quality of buffalo semen in relation to sperm DNA damage and ultrastructure, Reproductive Biology, 2017, 17: 42–50
Lee, Sung-Il et al. (2017) Evaluation of the shape, viability, stemness and osteogenic differentiation of cell spheroids formed from human gingiva‑derived stem cells and osteoprecursor cells, Exp Ther Med. 2017 Jun;13(6):3467-3473.
Dalia, Ali et al. (2016) Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells. Stem Cells Transl Med. 2016 Aug;5(8):1036-47.
Chen et al., PDGFB-based stem cell gene therapy increases bone strength in the mouse. PNAS, Jul 2015; 112: E3893 - E3900.
Dambatta et al., In vitro degradation and cell viability assessment of Zn–3Mg alloy for biodegradable bone implants. Journal of Engineering in Medicine, May 2015; 229: 335 - 342.
For more citations of this product click here