Product Name:	Alpha-Glucosidase Activity Assay Kit (BA0012)
SKU:	BA0012
Detection Method:	Colorimetric kinetic (OD 405 nm)
Detection Range:	2 to 250 U/L (20 µL sample); detection limit 2 U/L
Sample Type:	['Biological samples', 'Enzyme preparations']
Species Reactivity:	All
Assay Time:	20 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20°C
Shelf Life:	At least 6 months after receipt
Shipping:	Room Temperature

Alpha-glucosidase hydrolyses the terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. Alpha-glucosidase is needed by all animals to hydrolyse maltose to glucose for use as a food, and aberrant activities have been implicated in diseases such as diabetes and Pompe disease. This kit is designed to measure alpha-glucosidase activity directly in biological samples without pretreatment. The improved method utilises p-nitrophenyl-alpha-D-glucopyranoside that is hydrolysed specifically by alpha-glucosidase into a yellow coloured product (maximal absorbance at 405 nm). The rate of the reaction is directly proportional to the enzyme activity.

High sensitivity and wide linear range. Use 20 µL sample. The detection limit is 2 U/L, linear up to 250 U/L.
Homogeneous and simple procedure. Simple mix-and-measure procedure allows reliable quantitation of alpha-glucosidase activity within 20 minutes.
Robust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.

Direct Assays: alpha-glucosidase activity in biological samples.
Characterisation and Quality Control for alpha-glucosidase production.
Drug Discovery: high-throughput screen and evaluation of alpha-glucosidase inhibitors.

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Equilibrate reagents to room temperature. Prepare the Working Reagent by mixing, for each 96-well assay, 200 µL Assay Buffer and 8 µL alpha-NPG substrate (final 1.0 mM); fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature.
2	Prepare samples in 50 mM phosphate (pH 7.0) buffer or another suitable enzyme buffer. Avoid chemicals known to affect activity, including SH-containing reagents (dithiothreitol, 2-mercaptoethanol, glutathione), Ca2+, Cu2+, Fe3+/Fe2+, Hg2+, Mg2+, Ni2+, Zn2+, SDS, Triton X-100, Tween, digitonin, EDTA and Tris.
3	Transfer 20 µL distilled water to two wells of a clear-bottom 96-well plate, adding 200 µL H2O to one well and 200 µL Calibrator to the other (total volume 220 µL). Transfer 20 µL samples into other wells and transfer 200 µL Working Reagent to the sample wells only (final reaction volume 220 µL), then tap the plate briefly to mix.
4	Read OD405nm at t = 0, and again after 20 min on a plate reader.

Alpha-Glucosidase Activity = [(OD₂₀ - OD₀) / (OD_Calibrator - OD_H2O)] x 250 (U/L), where OD₂₀ and OD₀ are OD_405nm values of the sample at 20 and 0 min, and OD_Calibrator and OD_H2O are OD_405nm values of the Calibrator and water at 20 min. Unit definition: one unit of enzyme catalyses the hydrolysis of 1 µmole of substrate per min at pH 7.0.

Component	Quantity	Storage
Assay Buffer (pH 7.0)	24 mL	-20°C
alpha-NPG Substrate	1 mL	-20°C
Calibrator (equivalent to 250 U/L)	10 mL	-20°C

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Metabolism Assays

Alpha-Glucosidase Activity Assay Kit (BA0012)

Description