Total Soluble Sugar Quantification from Ethanolic Plant Extracts
Lynn Doran, Amanda P. De Souza
Abstract
Quantification of total soluble sugars (as glucose) in plant tissue extracts via the sulfuric phenol method adapted for 96 well plates.
Before start
Extract and purify total soluble sugars from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.
Steps
Glucose standard preparation
Prepare glucose standards in microcentrifuge by pipetting the appropriate amounts of 1 mg/mL Glucose standard and distilled water into each labeled tube.
| A | B | C |
|---|---|---|
| ug Glucose/50 ul-well (ug) | Amount 1 mg/mL Glucose (ul) | Amount distilled water (ul) |
| 5 | 20 | 180 |
| 10 | 40 | 160 |
| 15 | 60 | 140 |
| 20 | 80 | 120 |
| 25 | 100 | 100 |
Pipette 50 ul of each prepared glucose standard in triplicate into the assigned wells.
Sample preparation
Extract and purify total soluble sugars from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.
Pipette 10-30 ul of each sample extract in triplicate into the assigned wells. Record the amount of sample added.
Add distilled water to bring the total volume of each well to 50 ul. For example, if 10 ul of sample was used add 40 ul of distilled water.
Assay
Move the plate to the chemical fume hood.
Inside the hood, add 150uL of sulfuric acid to each well. Try to minimize the time between addition to first well and final well.
Immediately after the addition of sulfuric acid, add 30uL of phenol 5% in each well. Try to minimize the time between addition to first well and final well.
Incubate the plate by floating in a 90°Cwater bath for 0h 5m 0s.
Place plate on ice bath to cool until cool to the touch.
Once the plate is cool, read the absorbance at 490 nm on a UV-VIS spectrophotometer.
Additional Assay Plates
Do not shutoff the spectrophotometer lamp between plates. If the lamp remains on continuously, only one glucose standard curve is needed. If the lamp is shut off, a standard curve will need to be included.
Include a blank, 0 ug glucose standard (50 ul distilled water) on every plate.
Basic Calculations
Normalize each assay plates absorbances to zero using the 0 ug glucose standard per plate.
Calculate ug total soluble sugar as glucose for each sample using the averaged normalized standard curve absorbances for technical replicates and the averaged normalized sample absorbances for technical replicates.
Divide the ug total soluble sugar by the ul of total sample extract loaded.
Multiple the ug total soluble sugar per ul of total sample extract loaded by the total number of uls of distilled water the total soluble sugars were re-suspended in. If following the protocol, Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues., as written the total soluble sugar was resuspended in 1000 uls of distilled water.
Divide the ug total soluble sugar per 1 mL extract by the initial weight of ground tissue used in the ethanolic extraction (Step 1 of Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.) The final value reported will be ug Total Soluble Sugars (as glucose) per mg plant tissue.
