a-Glucosidase Assay Kit, BioAssay™

a-Glucosidase hydrolyzes the terminal, non-reducing 1,4-linked a-Dglucose residues with release of a-D-glucose. a-Glucosidase is needed by all animals to hydrolyze maltose to glucose for use as a food. Aberrant activities have been implicated in diseases such as diabetes and Pompe disease.

Simple, direct and automation-ready procedures for measuring a- glucosidase activity are becoming popular in Research and Drug Discovery. a-Glucosidase Assay Kit is designed to measure a-glucosidase activity directly in biological samples without pretreatment. The improved method utilizes p-nitrophenyl-a-Dglucopyranoside that is hydrolyzed specifically by a-glucosidase into a yellow colored product (maximal absorbance at 405nm). The rate of the reaction is directly proportional to the enzyme activity.

High sensitivity and wide linear range. Use 20ul 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 a-glucosidase activity within 20 minutes.
Robust and amenable to high throughput systems. All reagents are compatible with highthroughput liquid handling instruments.

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

Assay Buffer, pH 7.0, 1x24ml
a-NPG Substrate, 1x1ml
Calibratorl (equivalent to 250 U/L), 1x10ml

Store components at -20°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. 

Pipeting devices and accessories (e.g. multi-channel pipettor)
Clear bottom 96-well plates (e.g. Corning Costar)
plate reader

This assay is based on a kinetic reaction. Use of a multi-channel pipettor is recommended. Addition of Working Reagent to samples should be quick and mixing should be brief but thorough. Assays can be executed at room temperature or 37°C.

Reagent preparation: equilibrate reagents to room temperature. The Working Reagent is prepared by mixing for each 96-well assay, 200ul Assay Buffer and 8ul a-NPG substrate (final 1.0mM). Fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature.

Sample preparation: enzyme samples can be in 50mM phosphate buffer, pH 7.0 or in any other suitable enzyme buffer. The following chemicals are known to affect the enzyme activity and should be avoided. SH-containing reagents (e.g. dithiothreitol, 2-mercaptoethanol, glutathione), Ca2+, Cu2+, Fe3+/Fe2+, Hg2+, Mg2+, Ni2+, Zn2+, SDS, Triton X-100, Tween, digitonin, EDTA and Tris.

1. Transfer 20ul distilled water (dH2O) to two wells of a clear bottom 96-well plate. Add 200ul dH2O to one of these wells and 200ul Calibrator to the other well (total volume 220ul).
Transfer 20ul samples into other wells. Transfer 200ul Working Reagent to the sample wells only. The final reaction volume in the sample wells is 220ul. Tap plate briefly to mix.

2. Read OD405nm (t=0), and again after 20 min (t=20 min) on a plate reader.

3. Calculation: a-glucosidase activity of the sample (U/L) is

a-Glucosidase Activity = OD20 - OD0 x 250 (U/L)
OD(Calibrator) - OH(H2O)

OD20 and OD0 are OD405nm values of sample at 20 and 0 min, respectively. OD(calibrator) and OD(H2O) are OD405nm values of Calibrator and H2O at 20 min.
Unit definition: one unit of enzyme catalyzes the hydrolysis of 1uMole of substrate per min at pH 7.0.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.