This method allows researchers in the field of plant sciences and nutritional ecology to accurately measure concentrations of soluble proteins and digestible carbohydrates in plant tissue. The main advantage of these techniques is that they offer a quick and easy method for quantifying these two incredibly important macronutrients with high accuracy. These techniques have strong implications for the field of ecology because plant proteins and carbohydrates make up the base of terrestrial food webs.
Generally, individuals new to this technique may struggle because there are several steps and some techniques which are not commonly performed in a general-use lab. To begin, weigh out replicate samples of each tissue into labeled 1.5 milliliter microcentrifuge tubes. Next, record the precise mass of each sample.
Using a micropipetter, add 500 micro-liters of 0.1 molar sodium hydroxide to each tube. Close the lids tightly and sonic heat the tubes for 30 minutes. Next, place the tubes in a preheated hot-water bath for 15 minutes.
After this, centrifuge the tubes at 15, 000 G for 10 minutes. Pipette the supernatant into new, labeled microcentrifuge tubes, using a new pipette tip for each sample. Add 300 microliters of 0.1 molar sodium hydroxide to the pellet, and repeat the centrifugation for 10 minutes.
After this, remove the supernatant and transfer it to the tubes containing the supernatant from the first centrifugation. To neutralize the pH of the supernatant, add 11 microliters of 5.8 molar hydrochloric acid. Use litmus paper to confirm the pH is approximately seven.
Next, add 90 microliters of 100%trichloroacetic acid to each tube. Then incubate the tubes on ice for 30 minutes. Centrifuge the samples at 13, 000 G for 10 minutes at four degrees Celsius.
Carefully use a vacuum line and glass micropipette tip to remove the trichloroacetic acid. Be very careful not to disturb the pellet with the suction tip. Next, wash the pellet with 100 microliters of acetone cooled to 20 degrees Celsius, then allow the acetone to evaporate in a fume hood.
Dissolve the protein pellet with one milliliter of sodium hydroxide. Additional rounds of heating, vortexing and sonic heating may be required. Add 160 microliters of each IGG standard solution to a 96-well plate in triplicate, starting with the A1 position Next, in a new 1.5 milliliter tube add 50 microliters of each sample solution to 950 microliters of distilled water.
Then add 60 microliters of each diluted sample to the well plate in triplicate, starting with the H1 position. Add 100 microliters of distilled water to all of the blank and unknown sample wells. Using a multi-channel pipette, add 40 microliters of Coomassie Brilliant Blue G-250 protein dye to each well of the plate.
Using a needle, pop any bubbles present in the wells. After this, allow the plate to incubate at room temperature for five minutes. Then use a microplate spectrophotometer to record the absorbence values for each well at 595 nanometers.
First, weigh out replicates from each tissue sample into glass 15 milliliter tubes. Label the tubes and record the exact mass of each sample. Next, add one milliliter of 0.1 molar sulfuric acid to each tube and close the caps tightly.
Then place the tubes in a boiling-water bath for one hour. Transfer the tubes to a tepid water bath to cool. Then pour the contents of the tubes into labeled 1.5 milliliter microcentrifuge tubes.
After this centrifuge tubes at 15, 000 G for 10 minutes. Use a micro pipette to transfer the supernatant to new labeled 1.5 milliliter tubes. Using a pipette transfer 15 microliters of each unknown sample to its own test tube.
Then add 385 microliters of distilled water to each tube. In a fume hood add 400 microliters of 5%phenol to each standard and unknown sample test tube. Immediately thereafter add two milliliters of sulfuric acid to each tube.
Make sure to add the sulfuric acid to the surface of the solution. After incubating the tubes for 10 minutes, vortex the tubes. Then incubate the tube for an additional 30 minutes.
Transfer 800 microliter from each sample tube to three polystyrene 1.5 milliliter semi-microcuvettes. Then set the spectrophotometer to read at 490 nanometers and calibrated with a blank cuvette. Finally, run each cuvette through the spectrophotometer and record the absorbence.
Using this protocol, the soluble protein and digestible carbohydrate content of four different field and sweet corn tissues from three geographical regions were analyzed. Significant differences in soluble protein content were observed between the regions, thus necessitating separate analyses for each region. Across regions there were several differences in soluble protein and digestible carbohydrate content between tissues.
In Minnesota, tissues only varied in soluble protein content while North Carolina tissues varied in both soluble protein and digestible carbohydrates. Variability in tissue macronutrient content in Texas depended on the variety. This technique will allow researchers in the field of plant sciences and nutritional ecology to precisely measure soluble plant protein and digestible carbohydrates, rather than extrapolating from elemental measures.
Don't forget that working with liquid nitrogen, concentrated acids and bases, can be extremely hazardous. So always use precautions such as gloves, safety goggles, aprons and closed-toed shoes whenever working with these chemicals.
