Enzymatic Method for Determining Glucose and Sucrose (Glucose and Sucrose Assay)

Introduction

This assay protocol is suitable for the colorimetric/fluorometric detection of Glucose and Sucrose in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Glucose and Sucrose Assay Kit (MAK013). In this assay kit, glucose is oxidized via glucose oxidase resulting in a colorimetric (570 nm)/ fluorometric (λex = 535 nm/λem = 587 nm) product, proportional to the glucose present. To measure sucrose, invertase is added to the reaction to convert the sucrose to glucose and fructose. The free glucose can be subtracted from the total glucose to give the concentration of sucrose present. This kit has a linear range of detection between 0.2–1.0 nmole of glucose for the fluorometric assay and 2–10 nmoles of glucose for the colorimetric assay. 

Reagents

Glucose Assay Buffer                          25 mL
    Catalog Number MAK013A

Glucose Probe, in DMSO                    0.2mL
    Catalog Number MAK013B

Invertase                                            1 vL
    Catalog Number MAK013C

Glucose Enzyme Mix                          1 vL
    Catalog Number MAK013D

Sucrose Standard, 100 nmole/µL          0.1 mL   
    Catalog Number MAK013E

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – It is recommended to use black plates with clear bottoms (Catalog Number M5686 or equivalent) for fluorescence assays and clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.

Fluorescence or spectrophotometric multiwell plate reader


Preparation Instructions

Glucose Assay Buffer – Allow buffer to come to room temperature before use.

Glucose Probe – Ready-to-use as supplied. Allow the probe to come to room temperature before use. Store protected from light at –20 °C for use within 2 months.

Glucose Enzyme Mix and Invertase – Reconstitute each in 220 µL of Glucose Assay Buffer. Mix well by pipetting, then aliquot each and store protected from light at –20 °C. Use within 2 months of reconstitution and keep cold while in use.

Storage / Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

Sucrose Standards for Colorimetric Detection

Dilute 10 µL of the 100 nmole/µL Sucrose Standard Solution with 990 µL of Glucose Assay Buffer to prepare a 1 nmole/µL standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the 1 nmole/µL Sucrose standard solution into a 96 well plate, generating 0 (assay blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Glucose Assay Buffer to each well to bring the volume to 50 µL.

Sucrose Standards for Fluorometric Detection

Prepare a 1 nmole/µL standard solution as for the Colorimetric Assay. Dilute 20 µL of the 1 nmole/µL standard solution with 180 µL of Glucose Assay Buffer to generate a 0.1 nmole/µL standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the 0.1 nmole/µL Sucrose standard solution into a 96 well plate generating, 0 (assay blank), 0.2, 0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add Glucose Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation

Liquid samples can be measured directly. Bring samples to a final volume of 50 µL with Glucose Assay Buffer.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

For sucrose detection, set up duplicate wells for each sample in which glucose is measured. These wells will be used to determine total glucose. 

Assay Reaction

1.    Add 2 µL of Invertase to each of the sucrose samples and to the sucrose standards. Add 2 µL of Glucose Assay Buffer to the glucose sample. Incubate the plate at 37 °C for 30 minutes.

2.    Set up the Master Reaction Mix according to the scheme in Table 1. 50 µL of the Master Reaction Mix is required for each reaction (well).

Table 1.
Reaction Mixes

Reagent
Volume
Glucose Assay Buffer
46 µL
Glucose Probe
2 µL
Glucose Enzyme Mix
2 µL

3.    Add 50 µL of the Master Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at 37 °C. Protect the plate from light during the incubation.

4.    For colorimetric assays, measure the absorbance at 570 nm (A570). For fluorometric assays, measure fluorescence intensity (λex = 535/λem = 590 nm).

Results

Calculations

The background for the assays is the value obtained for the 0 (assay blank) Sucrose Standard. Correct for the background by subtracting the 0 (assay blank) value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate Sucrose standards to plot a standard curve.

Note: A new standard curve must be set up each time the assay is run.

Concentration of Glucose and Sucrose

        Sa/Sv = C

Sa = Amount of Glucose in unknown sample (µg) from standard curve
Sv = Sample volume (µL) added into the wells
C = Concentration of Glucose in sample

Sample Calculation

Amount of Glucose (Sa) = 5.84 nmole
    (from standard curve)
Sample volume (Sv) = 50 µL

Concentration of Glucose in sample

5.84 nmole/50 µL = 0.1168 nmole/µL

0.1168 nmole/µL x 180.6 ng/nmole=21.09 ng/µL

Sucrose = Total Glucose - Free Glucose

Materials

     

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

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