Dear Qian Wen Jun, 

Enzymatic Method for Determining Glucose-6-Phosphate (Glucose-6-Phosphate Assay)

Introduction

This assay protocol is suitable for the colorimetric detection of Glucose-6-Phosphate (G6P) in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Glucose-6-Phosphate Assay Kit (MAK014).  G6P concentration is determined by a coupled enzyme assay, which results in a colorimetric (450 nm) product, proportional to the G6P present. This kit has a linear range of detection between 0.2–1.0 nmole.

Reagents

Glucose-6-Phosphate Assay Buffer                    25 mL

    Catalog Number MAK014A

Glucose-6-Phosphate Enzyme Mix                    1 vL

    Catalog Number MAK014B

Glucose-6-Phosphate Substrate Mix                  1 vL

    Catalog Number MAK014C

Glucose-6-Phosphate Standard 10 µmole            1 vL

    Catalog Number MAK014D

Reagents and Equipment Required but Not Provided

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

Spectrophotometric multiwell plate reader

10 kDa Molecular Weight Cut-Off (MWCO) Spin Filter (Catalog Number Z706345 or equivalent)

Preparation Instructions

 Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Glucose-6-Phosphate Assay Buffer – Allow buffer to come to room temperature before use.

Glucose-6-Phosphate Enzyme Mix – Reconstitute in 220 µL of water. Mix well by pipetting, then aliquot and store at –20 °C. Use within 2 months of reconstitution.

Glucose-6-Phosphate Substrate Mix – Reconstitute in 220 µL of Glucose-6-Phosphate Assay Buffer. Mix well by pipetting, then aliquot and store at 4 °C. Use within 2 months of reconstitution.

Glucose-6-Phosphate Standard – Reconstitute in 100 µL of water to generate a 100 mM Standard Solution. Mix well by pipetting, then aliquot and store at –20 °C. Use within 2 months of reconstitution. 

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.

Glucose-6-Phosphate Standards for Colorimetric Detection

Dilute 10 µL of the 100 mM G6P Standard Solution with 990 µL of water to generate a 1 mM standard solution. Add 0, 2, 4, 6, 8, 10 µL of the 1 mM standard solution into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Glucose-6-Phosphate Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation

Liquid or solution samples may be assayed directly. If enzymes are present in the samples, samples should be deproteinized with a 10 kDa MWCO spin filter.

Tissue (10–100 mg) or cells (1–5 x 106) can be homogenized in 2–3 volumes of ice cold PBS or other buffer (pH 6.5–8). Centrifuge the samples at 13,000 x g for 10 minutes to remove insoluble material. Samples should be deproteinized with a 10 kDa MWCO spin filter.

Add 1–50 µL samples into duplicate wells of a 96 well plate. Bring samples to a final volume of 50 µL with Glucose-6-Phosphate Assay Buffer.

For unknown samples, it is suggested to test several sample volumes to make sure the readings are within the standard curve range.

Assay Reaction

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

Note: NADH or NADPH in samples will generate background readings. To remove the effect of NADH or NADPH background, a blank sample may be set up for each sample by omitting the Glucose-6-Phosphate Enzyme Mix. The blank readings can then be subtracted from the sample readings.

Table 1.

Reaction Mixes  

Reagent                      Blank Sample                 Samples & Standards 

G6P Assay Buffer        46 µL                                          46 µL

G6P Enzyme Mix          -                                                  2 µL

G6P Substrate Mix      2 µL                                             2 µL

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

 Measure the absorbance at 450 nm (A450).

Results

Calculations

The background for the assay is the value obtained for the 0 (blank) G6P standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings.

Use the values obtained from the appropriate G6P standards to plot a standard curve. The amount of G6P present in the samples may be determined from the standard curve.

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

Concentration of G6P

        Sa/Sv = C

Sa = Amount of G6P in unknown sample (nmole) from standard curve

Sv = Sample volume (µL) added into the wells

C = Concentration of G6P in sample

Sample Calculation

G6P Amount (Sa) = 5.84 nmole

Assay volume (Sv) = 50 µL

Concentration of G6P in sample

5.84 nmole/50 µL = 0.1169 nmole/µL

0.1169 nmole/µL x 260.14 ng/nmole=30.41 ng/µL

  http://www.sigmaaldrich.com/technical-documents/protocols/biology/glucose-6-phosphate-assay-kit-mak014.html

Hoping this will be helpful,

Rafik