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Product Details
| Cat # +Size | K422-500 |
|---|---|
| Size | 500 assays |
| Detection Method | Fluorescence (360/440) |
| Species Reactivity | Mammalian |
| Applications | The assay designed to measure ALP activity in serum and bio-samples with detection sensitivity ~1 µU |
| Features & Benefits | • Simple procedure; takes less than 1 hour • Fast and convenient • The kit is an ultra-sensitive, direct and HTS-ready fluorometric assay designed to measure AP activity in serum and other bio-samples. |
| Kit Components | • ALP Assay Buffer • MUP Substrate • 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. The change in alkaline phosphatase level and activity is associated with a lot of diseases in the liver and bones. Alkaline phosphatase is also a popular enzyme conjugated to secondary antibody in ELISA. In BioVision’s Alkaline Phosphatase Fluorimetric Assay Kit, ALP cleaves the phosphate group of the non-fluorescent 4-Methylumbelliferyl phosphate disodium salt (MUP) substrate resulting in an intense fluorescent signal (Ex/Em = 360nm/440nm). The kit is an ultra sensitive, simple, direct and HTS-ready assay designed to measure ALP activity in serum and bio-samples with detection sensitivity ~1 µU, more sensitive than colorimetric assays. The kit is suitable for both research and drug discovery.
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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.
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 -20degrees Celcius?
Samples homogenized in the assay buffer can be frozen in aliquots at -80°C until analysis.
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.
When the enzyme is resuspended and stored at 4C 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°C 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.
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 kits 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.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
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°C. 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 kits 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.
We have serum samples containing EDTA , but is not sure how much EDTA is in the samples. Can we use this kit with these samples?
In looking at multiple research papers we knowthat even small concs of EDTA will decrease the efficiency of this assay, if not completely inhibit it. Therefore, we would not recommend using such samples with this kit.
Khan et al., Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes Involves Inverse Regulation of Osteoblast Function by PGC-1α and Skeletal Muscle Atrogenes: AdipoR1 as a Potential Target for Reversing Diabetes-Induced Osteopenia. Diabetes, Jul 2015; 64: 2609 - 2623.
Holscher et al, Human Milk Oligosaccharides Influence Maturation of Human Intestinal Caco-2Bbe and HT-29 Cell Lines. J. Nutr., May 2014; 144: 586 - 591.
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