TITLE: Megazyme Sucrose D-Glucose Assay Kit (K-SUCGL)
AUTHORS: Likhithchandragiri  

[DESCRIPTION]
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. 




[STEPS]
SECTION: Preparing the Reagents
1. To prepare Solution 1: Acetate Buffer, dissolve the contents of Bottle 1 (20 mL) to 400 mL in distilled water. 

Store at 4 °C 

SECTION: Preparing the Reagents
2. 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

SECTION: Preparing the Reagents
3. 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. 

SECTION: Preparing the Reagents
4. 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 .

SECTION: Preparing the Reagents
5. 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). 
SECTION: Reactions:
6. 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. 

SECTION: Reactions:
8. 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.

SECTION: Reactions:
7. 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. 

SECTION: Reactions:
9. Incubate the 100 ug D-glucose standard, blanks, and samples in a water bath at 50 °C  for 20 min. 


SECTION: Absorbance Measurements
11. 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.

SECTION: Reactions:
10. 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 20 min.

Solutions that contain free D-glucose or sucrose that was inverted into D-glucose by the action of beta-fructosidase should turn red. 
SECTION: Absorbance Measurements
12. 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. 
SECTION: Calculations:
13. 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.