Similarly, in the fluorescence-based α-amylase and lipase assays, enzyme inhibition was underestimated when a substrate blank was omitted. In the absorbance-based α-glucosidase assay, enzyme inhibition was underestimated when a sample blank was omitted for the coloured plant extracts. In the three assays analysed here, using only a buffer blank underestimated the enzyme inhibitory potential of the test sample. Therefore, we have provided comprehensive, step-by-step protocols for α-glucosidase-, α-amylase- and lipase-inhibition assays that can be performed in 96-well format in a simple, fast, and resource-efficient manner with clear instructions for blank-correction and calculation of results. Although assays targeting the above enzymes are common in the literature, there is a scarcity of detailed published protocols. This is the first study to examine how different blank-correcting methods affect enzyme assay results. We investigated the influence of using different types of blanks on the final calculated activity/inhibition results for three enzymes of significance in diabetes and obesity α-glucosidase, α-amylase, and lipase. However, the blank-correction procedures reported in published literature are highly inconsistent. The appropriate use of blanks in enzyme assays is important for assay baseline-correction, and the correction of false signals associated with background matrix interferences. Enzyme assays have widespread applications in drug discovery from plants to natural products.
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