Excellent substrate for phosphatase based ELISA assays. It produces a soluble end product which is yellow in color and read at 405nm to 410nm. Both kinetic and end point determinations can be performed. Supplied as a 50X concentrate. Dual component system.
pNPP is the most conventional reagent employed for estimating alkaline phosphatase (AP) activity. It is usually provided in powder or tablet form and dissolved in buffer prior to use. Working concentrations range from 3.5-4.0mM when used for ELISA assays.
After completion of analyte binding to a solid phase and reaction with an AP labeled probe, diluted pNPP Solution is added. Hydrolysis of the phosphate ester liberates p-nitrophenol producing a yellow chromogen with an extinction coefficient of 1.83 x 10e4 mol-1cm-1 at 405nm.
The reaction is linear allowing kinetic assays. Endpoint analysis can be performed by stopping the reaction with strong base and reading the final color at 405nm. Transphorylating agents such as diethanolamine (DEA), 2-amino-2-methyl-1-propanol (AMP) or on some occasions 2-amino-2-methyl-1,3-propanediol (AMPD) are the prevalent buffers.
For maximum activity: dilute in N2600-15
Color and Clarity:
Colorless to slight yellow
Stability (4 degrees C):
To pass specifications
Absorbance:
To pass specifications
ELISA Performance:
To pass specifications
Also Available:
N2600: p-Nitrophenyl Phosphate (pNPP)
N2600-10: p-Nitrophenyl Phosphate (pNPP), 50X Concentrate, 0.19M/Liter
N2600-15: p-Nitrophenyl Phosphate (pNPP) Buffer, DEA
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