QuantiChromTM a-Glucosidase Assay Kit (DAGD-100)

α-GLUCOSIDASE hydrolyzes the terminal, non-reducing 1,4-linked α-Dglucose residues with release of α-D-glucose. α-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 α- glucosidase activity are becoming popular in Research and Drug Discovery. BioAssay Systems' QuantiChromTM α-Glucosidase Assay Kit is designed to measure α-glucosidase activity directly in biological samples without pretreatment. The improved method utilizes p-nitrophenyl-α-Dglucopyranoside that is hydrolyzed specifically by α-glucosidase into a yellow colored product (maximal absorbance at 405nm). The rate of the reaction is directly proportional to the enzyme activity.

KEY FEATURES

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 α-glucosidase activity within 20 minutes.

Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.

APPLICATIONS

Direct Assays: α-glucosidase activity in biological samples.

Characterization and Quality Control for α-glucosidase production.

Drug Discovery: high-throughput screen and evaluation of α-glucosidase inhibitors.

a-NPG Substrate: 1 mL

Calibrator: 10 mL (equivalent to 250 U/L)

Storage conditions. The kit is shipped at room temperature. Store all components at -20°C. Shelf life of at least 6 months.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

PROCEDURES. 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, 200 μL Assay Buffer and 8 μL α-NPG substrate (final 1.0 mM). 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 50 mM phosphate (pH 7.0) buffer 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.

Procedure using 96-well plate:

1. Transfer 20 μL distilled water (H2O) to two wells of a clear bottom 96-well plate. Add 200 μL H2O to one of these wells and 200 μL Calibrator to the other well (total volume 220 μL). Transfer 20 μL samples into other wells. Transfer 200 μL Working Reagent to the sample wells only. The final reaction volume in the sample wells is 220 μL. Tap plate briefly to mix.

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

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

[1]. Yamamoto, K. et al (2004). Val216 decides the substrate specificity of α-glucosidase in Saccharomyces cerevisiae. Eur. J. Biochem. 271 (16): 3414 - 3420

[2]. Ernst, H.A. et al (2005). Characterization of different crystal forms of the α-glucosidase MalA from Sulfolobus solfataricus. Acta Crystallogr Sect F Struct Biol Cryst Commun. 61(Pt 12): 1039–1042.

[3]. Kim, Y. et al. (2003). Inhibitory effect of pine extract on α-glucosidase activity and postprandial hyperglycemia. Nutrition 21(6): 756 – 761.

QuantiChromTM b-Glucosidase Assay Kit (DBGD-100)

Colorimetric Kinetic Determination of b-Glucosidase Activity

KEY FEATURES

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 b-glucosidase activity within 20 minutes.

Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.

APPLICATIONS

Direct Assays: b-glucosidase activity in biological samples.

Characterization and Quality Control for b-glucosidase production.

Drug Discovery: high-throughput screen for b-glucosidase modulators.

b-NPG Substrate: 1 mL

Calibrator: 10 mL (equivalent to 250 U/L)

Storage conditions. The kit is shipped at room temperature. Store all components at -20°C. Shelf life of at least 6 months.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

PROCEDURES. 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 Solution is prepared by mixing for each 96-well assay, 200 μL Assay Buffer and 8 μL b-NPG substrate (final 1.0 mM). 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 50 mM phosphate (pH 7.0) buffer 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, EDTA and Tris.

1. Transfer 20 μL distilled water (H2O) to two wells of a clear bottom 96-well plate. Add 200 μL H2O to one of these wells and 200 μL Calibrator to the other well (total volume 220 μL). Transfer 20 μL samples into other wells. Transfer 200 μL Working Reagent to the sample wells only. The final reaction volume in the sample wells is 220 μL. Tap plate briefly to mix.

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

4. Calculation: b-glucosidase activity of the sample (U/L) is

OD20 and OD0 are OD405nm values of sample at 20 and 0 min, respectively. ODCALIBRATOR and ODH2O are OD405nm values of Calibrator and H2O at 20 min.

Unit definition: one unit of enzyme catalyzes the hydrolysis of 1 μmole of substrate per min at pH 7.0.

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

[1]. Bhat, M.K. et al. (1993). Purification and characterization of an extracellular b-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity. Journal of General Microbiology 139: 2825-2832.

[2]. Kaur, J. et al (2007). Purification and characterization of b- glucosidase from Melanocarpus sp. Electronic J. Biotechn. 10: 260-270.

[3]. IWASHITA, K. et al. (1998) Purification and characterization of extracellular and cell wall bound
-glucosidases from Aspergillus kawachii. Bioscience, Biotechn. Biochem. 62 (10): 1938-1946.