Overview
Alkaline phosphatase can catalyze the hydrolysis of phosphate esters in an alkaline environment. Changes in the expression level and activity of alkaline phosphatase are related to many pathological conditions of the liver and bone. LiankeBio’s alkaline phosphatase activity detection kit uses 4-nitrophenyl phosphate (pNPP) as the substrate, which can conveniently, quickly and sensitively detect alkaline phosphatase activity in mammalian serum and other biological samples. .
Components
How to use
1. Reagent preparation: Take out all reagents (including test samples) and return to room temperature. Standard working solution: Take 15 μl of p-nitrophenol solution (10 mM) and dilute it with detection buffer to 0.3 ml, with a final concentration of 0.5 mM.
2. The detection plan is as follows. The amounts of standards used are 2, 4, 8, 16, 24, 32 and 40 μl respectively. It is recommended to set up two replicates. Normally, 50 μl is added to the sample. If the alkaline diluent phosphatase activity in the sample is too high, the amount of sample can be reduced or diluted appropriately. In order to eliminate the influence of the color of the colored sample itself, it is necessary to set up a sample control well, that is, add the sample and use the detection buffer instead of the chromogenic substrate control.
3. After adding all the samples, shake and mix on the microplate shaker for tens of seconds. Incubate at 37°C in the dark for 5 – 10 minutes. If the alkaline phosphatase activity in the sample to be tested is low, the incubation time can be appropriately extended to 30 minutes.
4. Add 100 μl stop solution to each well to stop the reaction.
5. Detect absorbance in the range of 405 nm or – 415 nm. If the measurement cannot be performed immediately, the measurement can be completed within a few hours, and the yellow color displayed will be stable within a few hours.
6. Result calculation: Subtract the OD value of the blank control well from the OD value of the standard well, and establish a standard curve between the calibrated OD value of the standard well and the corresponding p-nitrophenol concentration. Subtract the OD value of the sample control well from the OD value of the sample well, and substitute the calibrated sample OD value into the standard curve to calculate the p-nitrophenol concentration. Alkaline phosphatase activity (U/ml) = A/V/T. Where A is the amount of p-nitrophenol produced in the sample (μmol); V is the sample volume (ml); T is the reaction time (min).
7. Definition of alkaline phosphatase activity unit: the amount of alkaline phosphatase required to hydrolyze the pNPP chromogenic substrate to produce 1 μmol p-nitrophenol per minute in DEA buffer with pH 9.8 at 37°C. The amount is defined as an enzyme activity unit, also called a DEA enzyme activity unit. In glycine buffer at pH 9.6 at 25°C, the amount of alkaline phosphatase required to hydrolyze the pNPP chromogenic substrate to produce 1 μmol p-nitrophenol per minute is defined as one unit of enzyme activity, also known as an Glycine enzyme activity unit. One Glycine enzyme activity unit is equivalent to approximately 3 DEA enzyme activity units. This kit measures the DEA enzyme activity unit Rohm and Haas resin.
Notes
1. Please read the instructions carefully before using this product. This product is for scientific research only and is not for diagnostic use.
2. For your safety and health, please wear laboratory protective clothing, gloves, masks and other necessary protective equipment.
3. The sample cannot containThere are phosphatase inhibitors such as EDTA, oxalate, fluoride and citrate.
4. Avoid repeated freezing and thawing of all samples. It is recommended to aliquot and freeze for storage.
5. Serum samples usually need to be diluted 10 times, and other samples can be serially diluted to make the OD value fall within the detection range of the standard curve. The original lysis buffer or PBS or the detection buffer provided by this kit can be used for dilution.
References
Phosphatase Activity Assay Kit Product Introduction
