Description
Alkaline Phosphatase Assay Kit (Fluorometric) (BA0170) (BA0170)
The Alkaline Phosphatase Assay Kit (Fluorometric) (SKU: BA0170) provides a simple, sensitive and automation-ready method for the quantitative determination of alkaline phosphatase (ALP) activity in biological samples. Alkaline phosphatase catalyses the hydrolysis of phosphate esters in an alkaline environment and, in mammals, is found chiefly in the liver and bones, making its measurement valuable in research and drug discovery. This improved method employs 4-methylumbelliferyl phosphate, which is hydrolysed by ALP into the highly fluorescent product 4-methylumbelliferone. The rate of increase in fluorescence, measured at 360/450 nm, is directly proportional to the enzyme activity. Requiring only 10 uL of sample and offering a detection limit of 0.02 U/L, the kit delivers reliable results within 20 minutes. It is intended for research use only.
| Product Name: | Alkaline Phosphatase Assay Kit (Fluorometric) (BA0170) |
| SKU: | BA0170 |
| Detection Method: | Fluorimetric (lambda ex/em = 360/450 nm) |
| Detection Range: | Detection limit of 0.02 U/L (20 min reaction) |
| Sample Type: | Serum, plasma and cell culture media |
| Species Reactivity: | All |
| Assay Time: | 20 min |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 2 years after receipt |
| Shipping: | Room Temperature |
A homogeneous, mix-and-measure fluorimetric assay for the quantitative determination of alkaline phosphatase activity. The kit uses 4-methylumbelliferyl phosphate, which ALP hydrolyses to the highly fluorescent 4-methylumbelliferone; the rate of fluorescence increase (360/450 nm) is directly proportional to enzyme activity. It offers high sensitivity, a wide linear range and a detection limit of 0.02 U/L for a 20-minute reaction.
- High sensitivity and wide linear range; uses only 10 uL sample
- Detection limit of 0.02 U/L for a 20-minute reaction
- Homogeneous, simple mix-and-measure procedure completed within 20 minutes
- Robust and amenable to high-throughput screening
- All reagents compatible with high-throughput liquid-handling instruments
- Direct measurement of ALP activity in serum, plasma and other sources
- Characterisation and quality control of ALP production
- High-throughput screening for ALP modulators in drug discovery
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Thaw reagents to room temperature prior to use. Mix 5 uL of the provided 100x Standard (2 mM 4-methylumbelliferone) with 495 uL distilled water to obtain a 1x Standard (20 uM). Prepare the standard dilutions as shown in the table. |
| 2 | Transfer 10 uL of each Standard and each Sample to separate wells of a black flat-bottom plate. |
| 3 | Using a multi-channel pipettor, add 90 uL Reagent to all Standard and Sample wells. Quickly tap the plate to mix and incubate for a chosen period (e.g. 20 min) at a chosen temperature (e.g. 25C). |
| 4 | Read fluorescence intensity (lambda exc = 360 nm, lambda em = 450 nm) on a plate reader. |
Plot the RFU measured at 20 min for each Standard against the 4-methylumbelliferone concentration and determine the slope by linear regression. ALP activity = (F_SAMPLE - F_BLANK) / (Slope x t) x n (U/L), where F_SAMPLE and F_BLANK are the fluorescence values of the sample and the blank (no-standard well), t is the reaction time (e.g. 20 min) and n is the dilution factor. One unit (U) of ALP catalyses the conversion of 1 umole of 4-methylumbelliferyl phosphate to 4-methylumbelliferone per minute at pH 10.5 and 25C.
| Component | Quantity | Storage |
| Reagent (pH 10.5) | 14 mL | -20C |
| 100x Standard | 120 uL | -20C |
