PJK´s series of stabilized chemiluminescent 1,2-dioxetanes substrates can detect AP enzyme at the attogram level over a period of a few minutes.
- Blotting (Nitrocellulose Membrane, PDVF, Nylon)
- Reporter Gene Assays
- Quantitative PCR
Advantages of Chemiluminescent Substrates for AP:
- Formulated for two wavelength (450nm and 540nm) Safe- contains no organic solvents and no radioactive materials.
- Unsurpassed signal-up to 15 times brighter than leading commercial reagents.
- Low background-high signal to noise ratio allows increased signal without sacrifice.
- Steady glow-long lasting light emission without signal decay for up to four hours..
- Convenient-ready to use single bottle reagent requires no additional enhancers.
- Consistent results-reproducible results and ideal for automatic or manual systems.
- Very sensitive-less than one attogram of alkaline phosphatase has been detected.
- Wide dynamic range-5 to 6 log dynamic range standard curves over a broad range of times.
- Detection platforms-solution phase applications, membrane based applications and single tube or microplate format applications.
Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, alkaline optimum, AP) are found primarily in animal tissue and microorganism. AP used in enzyme immunoassays (EIA) are isolated from bovine intestinal mucosa or from E. coli. These enzymes have considerable differences in thier properties and should not be assayed under identical conditions. The bacterial enzyme has lower activity than the bovine intestinal enzyme.
Alkaline phosphatases hydrolyses numerous esters, such as those of primary and secondary alcohols, phenols and amines. A major reason for the popularity of alkaline phosphatases for EIA is ist absence from higher plants. The enzyme is abundant in animals and human tissues involved in nutrient transport and in developing tissues and secretor organs, but it is not found in significant amounts in muscle, connective tissue or cartilage. Some pathological conditions increase alkaline phosphatase activities in sera.
The enzyme transfers the phosphoryl residue via phosphoryl-enzyme intermediate, which can be repressed by organic phosphate. The comparative dtection limit of AP using fluorescence, time-resolved fluorescence and colorimetric techniques are 10-19M (6x104 molecules), 3x10-19M(1,8x105 molecules) and 5x10-17M (3x108 molecules), respectively. Stabilized 1,2-dioxetane substrates provide high signal, low background, wide dynamic range, rapid results and exellent reproducibility. These 1,2-dioxetanes provide substrates which are highly sensitive but can detect an enzyme concentration up to 10-21M (6x102 molecules of AP) in solution as well as on membrane.