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Protokoll
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1. Sample Preparations: Inhibitors of AP, like 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 for 3 minutes. Add test samples directly into 96-well plate, bring total volume to 110 μl with Assay Buffer. In order to avoid interference of other components in the sample, set a sample background control. Add the same amount of samples into separate wells, bring volume to 110 µl with Assay Buffer. Add 20 μl Stop Solution and mix well to terminate AP activity in the sample. 2. Dilute enough (1:10) 5 mM MUP substrate solution to 0.5 mM with Assay Buffer. Add 20 μl 0.5 mM MUP substrate solutions to each well containing the test samples and background controls. Mix well. Incubate the reaction for 30 min (or longer if AP activity in sample is low) at 25°C, protect from light. 3. Standard Curve: Dilute 10 μl of the 5 mM MUP solution with 990 μl Assay Buffer to generate 50 µM MUP standards. Add 0, 2, 4, 6, 8, 10 μl into 96-well plate in duplicate to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol/well MUP standard. Bring the final volume to 120 μl with Assay Buffer. Add 10 μl of AP enzyme solution to each well containing the MUP standard. Mix well. Incubate the reaction for 30 min at 25°C, protect from light. The ALP enzyme will convert MUP substrate to equal amount of fluorescent 4- Methylumbelliferone (4-MU). 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 prepare the sample background control in step 1), gently shake the plate. Measure fluorescence intensity at Ex/Em 360/440 nm using a fluorescence microtiter plate reader. 5. Calculation: Correct background by subtracting the value derived from the sample background controls. Plot 4-MU standard Curve. Apply sample readings to the standard curve to get the amount of 4-MU generated by AP sample. AP activity of the test samples can then be calculated: AP Activity = A/V/T (mU/ml) Where: A is amount of 4-MU generated by samples (in nmol). V is volume of sample added in the assay well (in ml). T is reaction time (in minutes). Unit Definition: 1 unit of AP is the amount of enzyme causing hydrolysis of 1 μmol of MUP per minute at 25 o C .
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