DBGD

							                                                       QuantiChrom T M -Glucosidase Assay Kit (DBGD -100)
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                                       Colorimetric Kinetic Determination of -Glucosidase Activity             3




DESCRIPTION                                                                                                        4. Calculation: -glucosidase activity of the sample (U/L) is
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3-GLUCOSIDASE is a glucosidase enzyme which acts upon 01->4 bonds                                                  =       OD20–OD0                     x   250 (U/L)
linking two glucose or glucose-substituted molecules (i.e., the disaccharide                                           OADCALIBRATOR – ODH2O
cellobiose). -Glucosidases are required by organisms (some fungi,
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bacteria, termites) for consumption of cellulose. Lysozyme is also a -                                     3       OD 20 and OD 0 are OD 4 05nm values of sample at 20 and 0 min,
glucosidase and is present in tears to prevent bacterial infection of the eye.                                                              respectively. ODCALIBRATOR and ODH2O are
In humans, lower activity of a -glucosidase isoform (lysosomal gluco-
                                                       3                                                                                    OD405nm values of Calibrator and H2O at 20 min.
cerebrosidase) has been related to Gaucher's disease and Parkinson's                                                  -Glucosidase Activity Unit definition: one unit of enzyme catalyzes the
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disease.                                                                                                                                    hydrolysis of 1 mole of substrate per min at pH
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Simple, direct and automation-ready procedures for measuring -                                         3
                                                                                                                   7.0.
glucosidase activity are becoming popular in Research and Drug Discovery.                                          MATERIALS REQUIRED, BUT NOT PROVIDED
BioAssay Systems' QuantiChromTM -Glucosidase Assay Kit is designed to
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                                                                                                                   Pipeting devices and accessories (e.g. multi-channel pipettor). Clear
measure -glucosidase activity directly in biological samples without
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                                                                                                                   bottom 96-well plates (e.g. Corning Costar) and plate reader.
pretreatment. The improved method utilize s p-nitrophenyl - -D-                                    3
                                                                                                                                0             5          10           15         20
glucopyranoside that is hydrolyzed specifically by -glucosidase into a             3
                                                                                                                                                                  Time (min)
yellow colored product (maximal absorbance at 405nm). The rate of the
reaction is directly proportional to the enzyme activity.

KEY FEATURES                                                                                                                   0.5
High sensitivity and wide linear range. Use 20 L sample. The detection
limit is 2 U/L, linear up to 250 U/L.
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                                                                                                                                           -Glucosidase
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Homogeneous and simple procedure. Simple “mix-and-measure”                                                                     0.4         (0 to 250 U/L)
procedure allows reliable quantitation of -glucosidase activity within 20
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minutes.                                                                                                                       0.3
Robust and amenable to HTS. All reagents are compatible with high-
throughput liquid handling instruments.
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APPLICATIONS
Direct Assays: -glucosidase activity in biological samples.
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Characterization and Quality Control for -glucosidase production.      3
                                                                                                                               0.1
Drug Discovery: high-throughput screen for -glucosidase modulators.        3




KIT CONTENTS (100 tests in 96-well plates)                                                                                       0
     Assay Buffer: 12 mL (pH 7.0)                                                                                                                      [Enyzme Activity], U/L
     -NPG Substrate: 1 mL
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     Calibrator: 10 mL (equivalent to 250 U/L)                                                                              Kinetics of -glucosidase reaction in 96-well plate assay
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Storage conditions. The kit is shipped at room temperature. Store all
components at -20°C. Shelf life of at least 6 months.                                                                            0.5
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents. Please                                                         0.4               -Glucosidase
                                                                                                                                                   3


refer to Material Safety Data Sheet for detailed information.
PROCEDURES. This assay is based on a kinetic reaction. Use of a                                                                  0.3
multi-channel pipettor is recommended. Addition of Working Reagent to
samples should be quick and mixing should be brief but thorough. Assays                                                          0.2
can be executed at room temperature or 37 C.                   °                                                                                                        R2 = 0.998
Reagent preparation: equilibrate reagents to room temperature. The                                                               0.1
Working Solution is prepared by mixing for each 96-well assay, 200 L                                   R


Assay Buffer and 8 L -NPG substrate (final 1.0 mM). Fresh
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                                                                                                                                 0.0
reconstitution is recommended, although the Working Solution is stable
for at least one day at room temperature.                                                                                                  0                50 100 150 200 250
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                                         LITERATURE
known to affect the enzyme activity and should be avoided. SH-containing                                            [1]. Bhat, M.K. et al. (1993). Purification and characterization of an
reagents (e.g. dithiothreitol, 2-mercaptoethanol, glutathione), Ca2+, Cu2+,                                        extracellular p-glucosidase from the thermophilic fungus Sporotrichum
Fe3+/Fe2+, Hg2+, Mg2+, Ni2+, Zn2+, SDS, EDTA and Tris.                                                             thermophile and its influence on cellulase activity. Journal of General
                                                                                                                   Microbiology 139: 2825-2832.
Procedure using 96-well plate:
1. Transfer 20 L distilled water (H2O) to two wells of a clear bottom 96-well
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                                                                                                                    [2]. Kaur, J. et al (2007). Purification and characterization of 13 -
   plate. Add 200 L H2O to one of these wells and 200 L Calibrator to the
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                                                                                                                   glucosidase from Melanocarpus sp. Electronic J. Biotechn. 10: 260-270.
   other well (total volume 220 L).                R                                                                [3]. IWASHITA, K. et al. (1998) Purification and characterization of
         Transfer 20 L samples into other wells. Transfer 200 L Working
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                                                                                                                   extracellular and cell wall bound p-glucosidases from Aspergillus
         Reagent to the sample wells only. The final reaction volume in the                                        kawachii. Bioscience, Biotechn. Biochem. 62 (10): 1938-1946.
         sample wells is 220 L. Tap plate briefly to mix.
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    2. Read OD405nm (t = 0), and again after 20 min (t = 20 min) on a plate reader.