What Is the Detailed Experimental Protocol for the α-Glucosidase Activity Assay Following Polysaccharide Extraction?
To evaluate α-glucosidase activity after polysaccharide extraction, the following stepwise experimental protocol is employed:
Preparation of the Reaction Mixture
Prepare a solution of the α-glucosidase substrate, such as p-nitrophenyl-α-D-glucopyranoside (PNPG). Combine the substrate solution with a measured amount of the polysaccharide sample.
Addition of the Enzyme Solution
Introduce a defined volume of α-glucosidase solution into the reaction mixture.
Incubation
Incubate the reaction mixture under controlled temperature and pH conditions for a predetermined duration to allow enzymatic hydrolysis to proceed.
Termination of the Reaction
At the end of the incubation period, terminate the reaction by adding a stop solution, such as sodium carbonate (Na₂CO₃).
Measurement of the Reaction Product
Quantify the formation of p-nitrophenol (PNP), the hydrolytic product of PNPG, using UV–Vis spectrophotometry at a wavelength of 405 nm.
Data Analysis
Calculate α-glucosidase activity, typically expressed as the amount of PNP released per unit time (e.g., μmol/min).
Note: The above protocol outlines the fundamental steps for α-glucosidase activity determination. Reaction parameters—including temperature, pH, incubation time, and reagent concentrations—should be optimized based on experimental objectives and the nature of the polysaccharide sample.
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