Oct 18, 2021
Megazyme Sucrose D-Glucose Assay Kit (K-SUCGL)
- 1IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: Likhith Chandragiri 2021. Megazyme Sucrose D-Glucose Assay Kit (K-SUCGL). protocols.io https://dx.doi.org/10.17504/protocols.io.by5xpy7n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 18, 2021
Last Modified: October 18, 2021
Protocol Integer ID: 54167
Abstract
Protocol for a colorimetric assay kit to determine sucrose and D-glucose concentrations by Megazyme.
Guidelines
1. Store all reagent solutions as per the instructions given in the protocol. Keep the 1.65 mL working stocks for Solution 4 and Solution 4 covered in aluminium foil at all times.
2. Thaw the frozen Solution 2 (beta-fructosidase) and 1.65 mL working stocks for Solution 4 (GOPOD reagent buffer) in a 4°C refrigerator before use.
Materials
Kit contents:
Bottle 1: Acetate buffer (20 mL, pH 4.6). Store at 4°C.
Bottle 2: Beta-fructosidase (invertase) and sodium azide preservative (5 mL). Store at 4°C.
Bottle 3: GOPOD Reagent Buffer, p-hydroxybenzoic acid and sodium azide (50 mL. pH 7.4). Store at 4°C.
Bottle 4: GOPOD Reagent Enzymes - Glucose oxidase, peroxidase and 4-aminoantipyrine. Freeze-dried powder. Store at -10°C.
Bottle 5: D-Glucose standard solution (5 mL, 1mg/mL) in benzoic acid. Store at room temperature.
Bottle 6: Control flour sample. Store at room temperature.
Apparatus:
Test tubes
Microcentrifuge tubes
Pipettes
Water bath
Spectrophotometer or 96 well plate reader
Safety warnings
Use gloves while handling Bottle 2, Bottle 3, Solution 2, Solution 3 and Solution 4 as they contain sodium azide, which is toxic
Preparing the Reagents
Preparing the Reagents
To prepare Solution 1: Acetate Buffer, dissolve the contents of Bottle 1 (20 mL ) to 400 mL in distilled water.
Store at 4 °C
To prepare Solution 2: Beta-fructosidase, dissolve 1 mL of solution from Bottle 2 (20 mL ) to 10 mL in distilled water.
For convenience, prepare ten 1 mL aliquots of this. Store them at -20 °C
To prepare Solution 3: GOPOD Reagent Buffer, dissolve 5 mL of solution from Bottle 3 (50 mL ) to 100 mL in distilled water.
Divide Solution 3 into two portions of 20 mL and 80 mL .
Solution 3 must be used immediately and can't be stored.
To prepare Solution 4: GOPOD Reagent, dissolve all of the contents of Bottle 4 into the 20 mL portion of Solution 3.
For convenience, divide this into twelve aliquots of 1.65 mL each as working stocks.
Cover completely in aluminium foil and store at -20 °C .
Add 1.632 mL from the 1.65 mL aliquots of the working stock to the 80 mL portion of Solution 3. This is your Solution 4 (GOPOD Reagent).
Cover completely in aluminum foil and store at 4 °C .
To prepare fresh Solution 4 (GOPOD Reagent) in the future from the working stock aliquots:
First prepare 80 mL of fresh Solution 3 (GOPOD Reagent Buffer) by dissolving 4 mL of the solution from Bottle 4 to 80 mL in distilled water.
Add 1.632 mL from the 1.65 mL aliquots of the working stock to this 80 mL of freshly prepared Solution 3
Verify that the absorbance of Solution 4 at 510 nm against a distilled water blank is <0.05,
Note: Solution 3 cannot be stored and must be prepared freshly to make a fresh batch of Solution 4 (GOPOD Reagent).
Reactions:
Reactions:
20m
20m
100ug D-Glucose standard:
Take 0.1 mL of the solution from Bottle 5 in a test tube.
Add 0.3 mL of distilled water.
Sample:
Dilute your sample according to the directions in the kit instructions or by any factor that would bring your expected sucrose+glucose concentration to the range of 0.05 g/L to 1 g/L (as per our standardization curve).
Take 0.1 mL of the diluted sample in a 1 mL MCT, and add 0.1 mL of Solution 1 (Acetate buffer). Denote as Solution A.
Take 0.1 mL of the diluted sample in another 1 mL MCT, and add 0.1 mL of Solution 2 (Beta-fructosidase). Denote as Solution B.
Blanks:
In a 1 mL microcentrifuge tube (MCT), add 0.1 mL of distilled water and add 0.1 mL of Solution 1 (Acetate buffer). This is the blank for Solution A.
In another 1 mL MCT, add 0.1 mL of distilled water and add 0.1 mL of Solution 2 (Beta-fructosidase). This is the blank for Solution B.
Incubate the 100 ug D-glucose standard, blanks, and samples in a water bath at 50 °C for 00:20:00 .
20m
Add 1.5 mL of Solution 4 (GOPOD Reagent) to the 100 ug D-glucose standard, blanks, and sample A and B solutions.
Incubate in a water bath at 50 °C for 00:20:00 .
Solutions that contain free D-glucose or sucrose that was inverted into D-glucose by the action of beta-fructosidase should turn red.
20m
Absorbance Measurements
Absorbance Measurements
Pipette 0.3 mL from each of the reaction mixtures (standard, blanks, sample A and B solutions) into the wells of a 96 well plate.
Measure the absorbance of the reaction mixtures at 510 nm in a 96 well plate reader.
Otherwise, a regular cuvette and spectrophotometer can be used to measure absorbance.
Calculations:
Calculations:
Define ∆A as the absorbance of Solution A of the sample against the acetate buffer blank and ∆B as the absorbance of Solution B of the sample against the beta-fructosidase blank.
Define factor to convert from absorbance to ug for 100ug of D-glucose as F.
F = 100/absorbance of the 100ug D-glucose standard.
Define D as the factory by which the sample was diluted. For example, if 1 mL of the sample was diluted to 100mL with distilled water, D = 100.
Sucrose concentration is given by:
(∆B-∆A) x F x D x 0.0095
See the Megazyme kit instructions for the derivation of the above formula.