TITLE: Total carbohydrate content for coral samples
AUTHORS: Keyla Plichon, Paola Furla

[DESCRIPTION]
This protocol aims to quantify total carbohydrate content from a cytosolic extraction. It has been optimized for carbohydrate analysis from coral species. Here, the cytosolic extraction containing proteins and carbohydrates was extracted using the "Cytosolic content extraction from coral samples" method (doi). The quantification protocol is adapted from the phenol sulfuric method in a microplate format described by Masuko et al. (2005). The carbohydrate content is normalized by the animal biomass determined by protein quantification using the Bradford method. 

[BEFORE_START]
Determine protein concentration for each sample.

[GUIDELINES]
The protocol can be completed within one hour.

[STEPS]
SECTION: Carbohydrate quantification in a 96-well plate
5. Carbohydrate standard for colorimetric detection.
Add 0, 1, 2, 4, 6, 8, 10, and 12 μL of the glucose stock solution directly into the 96-well plate. Bring the volume to 30 μL. Each well will, therefore, contain 0 (blank), 2, 4, 8, 12, 16, 20, and 24 μg.
SECTION: Carbohydrate quantification in a 96-well plate
6. Sample preparation.
Prepare a dilution of the homogenate in which the protein amount is known. This protein content will be used for normalization. For Pocillopora sp. and Porites sp. samples, the protein final concentration in each well was 0.1 μg/μL.

Add 30 μL of sample in each well in triplicates (one well will be used as blank for each sample). For the first assay, It may be necessary to prepare several dilutions of unknown samples to ensure that readings are within the standard range.  Each plate can contain 24 samples.

 

SECTION: Prepare stock solutions and set up
1. Prepare a stock of standard solution.
Prepare a standard stock solution of D-glucose with a final concentration of 2 g/L. Store it at 4°C in the dark. This standard solution should be prepared with the buffer solution used for tissue homogenate. Porro et al. (2023) describe the buffer used in this protocol.
SECTION: Prepare stock solutions and set up
2. Prepare a stock solution of phenol 5%.
Dilute the phenol solution in MilliQ water and store it at 4°C.
SECTION: Prepare stock solutions and set up
3. Prepare a stock of concentrated (100%) sulfuric acid.
Keep it at room temperature.


SECTION: Carbohydrate quantification in a 96-well plate
7. Add concentrated sulfuric acid.
Add 150 μL of the concentrated sulfuric acid to each well and mix by pipetting. Cover the plate and protect it from light. Incubate the reaction for 15 min in a water bath at 90°C.
SECTION: Carbohydrate quantification in a 96-well plate
8. Add the MilliQ (blanks) and reagent (phenol).
Remove the plate from the water bath and, at room temperature, add 30 μL of Milli-Q to the blank wells and 5% phenol solution to the duplicate wells, taking care to mix well. Incubate the plate for 5 min at room temperature.
SECTION: Carbohydrate quantification in a 96-well plate
9. Measure the absorbance.
Mix the well content before measuring absorbance at 490 nm. This step can be performed using a multi-pipette or a plate shaker. Read absorbance at 490 nm.
SECTION: Carbohydrate quantification in a 96-well plate
10. Calculations.
Correct the background by subtracting the blank values from all readings. Construct the standard curve by performing the mean of each concentration. 

Use the standard curve equation to determine the carbohydrate content in 30 μL. Normalize by the amount of protein contained in the same volume. 

Results are then expressed in μg of carbohydrate/μg of protein.


SECTION: Prepare stock solutions and set up
4. Set up the water bath.
Place the water bath under the fume hood and make sure the water is at 90°C. If you do not have a 96-well plate water bath, you can use a heating plate with a glass water container. Ensure enough water to heat the wells evenly without the plate sinking (see Figure 1.)