Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is ...Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.展开更多
Saturated row-column designs are studied to eliminate positional effects in primary high- throughput screening experiments. Compared to designs currently used in practice, all compounds in the designs considered are c...Saturated row-column designs are studied to eliminate positional effects in primary high- throughput screening experiments. Compared to designs currently used in practice, all compounds in the designs considered are comparable within each microplate in spite of the existence of row and column effects. The designs considered in this paper also have the maximum number of compounds arranged in each microplate. Two statistical methods are used to choose leading compounds in the designs considered. These two methods take full advantages of effect sparsity in primary screening. Simulation studies are carried out to compare the two statistical methods with two ad hoc methods in selecting active compounds. A method that maintains balanced false positives and false negatives is recommended.展开更多
基金Project(31000350)supported by the National Natural Science Foundation of China
文摘Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.
文摘Saturated row-column designs are studied to eliminate positional effects in primary high- throughput screening experiments. Compared to designs currently used in practice, all compounds in the designs considered are comparable within each microplate in spite of the existence of row and column effects. The designs considered in this paper also have the maximum number of compounds arranged in each microplate. Two statistical methods are used to choose leading compounds in the designs considered. These two methods take full advantages of effect sparsity in primary screening. Simulation studies are carried out to compare the two statistical methods with two ad hoc methods in selecting active compounds. A method that maintains balanced false positives and false negatives is recommended.
