Method for screening and detecting phosphatidase C
A phospholipase and phosphatase technology, applied in the field of detection of phospholipase C and screening, can solve the problems of high cost of substrate synthesis, complicated and unanalyzable results, and achieve the effects of convenient mass screening, simple operation and low detection cost.
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Embodiment 1
[0147] Embodiment 1, standard curve preparation
[0148] Take 0.1431g of the dried dipotassium hydrogen phosphate reagent powder, dissolve it in deionized water, dilute to 100mL, and prepare 1mg / mL PO 4 3- dipotassium hydrogen phosphate solution.
[0149] From 1mg / mL PO 4 3- Take out 0, 2, 5, 8, 10, 15, 20, and 50 μL of dipotassium hydrogen phosphate solution, add deionized water to make up to 940 μL, then add 20 μL of 10% (w / v) ascorbic acid solution, shake well for 30 seconds Add 40 μL of 2.5% (w / v) ammonium molybdate solution (solvent is 30% sulfuric acid solution), and finally make up to 1 mL, the final PO 4 3- Concentrations are 0, 2, 5, 8, 10, 15, 20, 50 μg / mL. Three replicate samples were made in parallel for each concentration point.
[0150] After bathing in water at 37°C for 10 minutes, the absorbance value was detected at an absorbance of 700nm, and the obtained data was linearly fitted to obtain the standard curve for the detection of phosphomolybdenum blue....
Embodiment 2
[0153] Prepare 10 μg / μL PO 4 3- Phosphorylcholine solution, respectively take 0, 5, 10, 20, 40, 50, 80 μg PO 4 3- The phosphorylcholine solution in the total system 100μL, the reaction system also contains 50mM Tris-Cl (pH9.0), 10mM MgCl 2 , add alkaline phosphatase to 10U / mL.
[0154] Bath at 37°C for 30 minutes, add to 840 μL of deionized water, then add 20 μL of 10% (w / v) ascorbic acid solution, shake well for 30 seconds, add 40 μL of 2.5% (w / v) ammonium molybdate solution, and finally dilute to 1mL.
[0155] Water bath at 37°C for 10 minutes, detect the absorbance value at an absorbance of 700nm, and perform linear regression on the obtained absorbance value to obtain the corresponding PO 4 3- The quality of the resulting product PO 4 3- Mass number divided by the initially added substrate PO 4 3- Mass number, and then divided by the reaction time, the same amount of alkaline phosphatase in different substrates PO 4 3- The reaction rate at the mass number. by ...
Embodiment 3
[0158] Alkaline phosphatase was serially diluted, and 0, 0.05, 0.1, 0.2, 0.4, 0.5, 1.0, 1.5U were taken in 100μL system, and the reaction system also contained 50mM Tris-Cl (pH9.0), 10mM MgCl 2 , 50 μg PO 4 3- of phosphorylcholine solution.
[0159] Water bath at 37°C for 30 minutes, carry out color development and detection by molybdenum blue method according to the method of Example 2, and obtain the corresponding PO after performing linear regression on the obtained absorbance value 4 3- The quality of the resulting product PO 4 3- The mass number is divided by the 50 μg substrate PO initially added 4 3- , and then multiplied by 100% to get the reaction product PO 4 3- generation rate. Take the alkaline phosphatase concentration as the abscissa, and take the reaction product formation rate as the ordinate to obtain the relationship between the two. The results are as follows image 3 shown.
[0160] image 3 The results show that: with the increase of the amount...
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