Total Starch (as Glucose) Quantification by NZYtech GOD-POD Method
Lynn Doran, Amanda P. De Souza
Abstract
Total starch was previously extracted from plant tissue and enzymatically digested to D-Glucose. This protocol is based on the NZYtech GOD-POD kit's colorimetric determination of D-Glucose and includes basic calculations to convert D-Glucose back to total starch in the original tissue.
Before start
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues. Enzymatically digest starch to glucose per Total Starch Enzymatic Digestion.
Steps
Prepare Glucose Standards
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 |
|---|---|---|
| A | B | C |
| ug Glucose/20 ul-well (ug) | Amount 1 mg/mL Glucose (ul) | Amount distilled water (ul) |
| 0 | 0 | 80 |
| 2.5 | 10 | 70 |
| 5 | 20 | 60 |
| 7.5 | 30 | 50 |
| 10 | 40 | 40 |
| 12.5 | 50 | 30 |
Pipette 20 ul of each prepared glucose standard in triplicate into the assigned wells.
Sample Preparation
Extract and digest total starch from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues. and Total Starch Enzymatic Digestion.
Pipette 5-20 ul of each sample extract in triplicate into the assigned wells. Record the amount of sample added.
Assay
Prepare GOD-POD reagent per the instructions in the NZYtech GOD-POD protocol. Follow shelf life and storage requirements as listed in the NZYtech GOD-POD protocol.
Add 300 uL of prepared GOD-POD in each well using the multi-channel.
Incubate the plates at 45°C for 20 min.
Read the absorbance at 510 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 (20 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 starch 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 starch by the ul of total sample extract loaded.
Multiply the ug total starch per ul of total sample extract loaded by the total number of uls of buffer the total starch was re-suspended in. If following the protocol, Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues. and Total Starch Enzymatic Digestion. , as written the total starch was resuspended in 2000 uls of buffer.
Divide the ug total starch per 2 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 Starch (as glucose) per mg plant tissue.
