PNPP Microwell Alkaline Phosphatase (AP) Stabilized Substrate One Component

Store at temperatures between 2-8ºC. The substrate performance has been shown to be within specifications following 5 freeze-thaw cycles, however freezing is not recommended. The substrate should be protected from direct light by storing in amber bottles. Only high quality glass and plastic products should be used for storing aliquots.

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Stable for a minimum of 48 months from the manufactured date when properly stored between 2-8ºC.

pNPP Alkaline Phosphatase Microwell Substrate (p-nitrophenylphosphate) is a soluble substrate used with the enzyme alkaline phosphatase designed for various qualitative or quantitative immunoassays but not recommended for membrane or immunohistochemical applications where a precipitating reaction product is required. The substrate is supplied as a one component ready to use solution. Initially the substrate should be colorless to pale yellow in appearance. When this substrate system is reacted with alkaline phosphatase, a soluble, yellow product is obtained.

pNPP Alkaline Phosphatase Microwell Substrate is a ready to use solution that needs no preparation or dilution. Pour estimated amount of substrate into a suitable high quality plastic reservoir to avoid contamination of the bulk solution. It is recommended that you allow the substrate solution to equilibrate to room temperature before use. While the pNPP solution is equilibrating, wash the microplates thoroughly to remove excess alkaline phosphatase labeled conjugates. Washing the plates at least four times is recommended to minimize background noise.

For immunoassay microwell applications 100 – 200 μl of substrate solution is added to each well producing a soluble, yellow product. For best results, sample OD values should be monitored and read before absorbance values exceed 2.0 OD units. The substrate reaction can be stopped using 50 μl of pNPP Stop Solution (Leinco Prod. No.: P227) or 50 μl of 3N NaOH per 200 μL of substrate. Sample OD values are read, with or without the addition of stop solution, in the 405-420nm range. Dilution of the substrate is not recommended. To reduce the intensity of a reaction, it is recommended that the antibodies or conjugates be diluted. This substrate is light sensitive and should be protected from direct sunlight or UV sources.