|
Protokoll
|
1. Sample Preparations: Inhibitors of AP, such as tartrate, fluoride, EDTA, oxalate, and citrate, should be avoided in sample preparation. Serum, plasma, urine, semen, and cell culture media can be assayed directly. Cells (1×10 5 ) or tissue (~10 mg) can be homogenized in 100 μl Assay Buffer, centrifuge to remove insoluble material at 13,000g, 3 minutes. Add test samples directly into 96-well plate, bring total volume to 80 μl with Assay Buffer. If samples contain color, it may interfere with O.D. 405 nm readings, use a sample background control. Add the same amount of sample into separate wells, bring volume to 80 µl. Add 20 μl stop solution and mix well to terminate AP activity in the sample. 2. Add 50 μl of p NPP Substrate Solution to each well containing the test samples and background controls. Mix well. Incubate the reaction for 60 min at 25°C, protect from light. 3. Standard Curve: Dilute 40 μl of the 5 mM p NPP solution with 160 μl Assay Buffer to generate 1 mM p NPP standard. Add 0, 4, 8, 12, 16, 20 μl into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nmol/well p NPP standard. Bring the final volume to 120 μl with Assay Buffer. Add 10 μl of AP enzyme solution to each well containing the p NPP standard. Mix well. The AP enzyme will convert p NPP substrate to an equal amount of colored p -Nitrophenol ( p NP). Incubate the reaction for 60 min at 25°C, protect from light. 4. Stop all reactions by adding 20 μl Stop Solution into each standard and sample reaction except the sample background control reaction (since 20 μl Stop Solution has been added into the background control when the sample was prepared in step 1), gently shake the plate. Measure O.D. 405 nm in a micro-plate reader. 5. Calculation: Correct background by subtracting the value derived from the 0 standard from all the standards, samples and sample background controls (The background reading can be significant and must be subtracted from sample readings). Plot p NP standard Curve. Apply sample readings to the standard curve to get the amount of p NP generated by AP sample. AP activity of the test samples can then be calculated: AP activity (U/ml) = A/V/T Where A is amount of p NP generated by samples (in μmol). V is volume of sample added in the assay well (in ml). T is reaction time (in minutes) Unit Definition: One unit of AP is the amount of enzyme causing the hydrolysis of one micromole of p NPP to p NP per minute at pH 5.0 and 25°C.
|
