Soluble Protein Assay to Quantify Soluble Protein Content in Plant Tissues

1. Plant Collection and Processing

1. Collect and process plant samples
   1. After collecting plant samples, flash-freeze samples by dipping plant material into liquid nitrogen with forceps and store at -80 °C. If the plant samples collected are too large to flash-freeze, quickly cool the samples using dry ice and transfer to a -80 °C freezer as soon as possible. The macronutrient content of plant material can change after tissues are separated from the plant, so it is important to freeze plant samples as soon as possible after collection.
      CAUTION: Liquid nitrogen can cause severe frostbite when in contact with skin. Please make sure to transport liquid nitrogen in approved containers and wear proper PPE during its handling, including cryo-gloves, eye goggles, face shield, apron, and a lab coat.
   2. Lyophilize plant material using a freeze-dryer to ensure that the tissues are metabolically inactive while water is being removed. Dry until the mass of the sample stabilizes to ensure all water has been removed.
   3. Once samples are dry, grind into a fine powder using a grinder or mill. Store samples in a desiccating cabinet until analysis.

2. Soluble Protein Assay

1. Sample Preparation
   1. Weigh out replicate samples of each tissue, approximately 20 mg each, into 1.5 mL polystyrene microcentrifuge tubes and label tubes. These samples will subsequently be referred to as unknown samples. Record the exact mass of each sample, as this information will be required to calculate the %soluble protein in each unknown sample. Use microcentrifuge tubes with locking lids, as non-locking lids may open during the subsequent heating step, resulting in loss of sample solution.
2. Solubilize and isolate unknown sample proteins
   1. Using a micro-pipettor, add 500 µL of 0.1 M NaOH to each tube. Close lids tightly and sonicate for 30 minutes. Preheat a hot water bath 90 °C.
      CAUTION: Sodium hydroxide is a strong base and can cause burns when in contact with skin. Although 0.1 M NaOH represents a low concentration, wear protective gloves to avoid skin irritation.
   2. Place tubes in a rack in the hot water bath for 15 minutes.
   3. Centrifuge tubes at 15,000 x g for 10 minutes. Pipette the supernatant liquid into new labeled microcentrifuge tubes, using a new pipette tip for each sample. Add 300 µL of 0.1 M NaOH to the tube containing the pellet and centrifuge again at 15,000 x g for 10 minutes. Again, collect the supernatant liquid and combine it with the other supernatant fluid from the same sample. This is a potential stopping point, and samples can be stored at 4 °C overnight if needed.
3. Precipitate plant proteins
   1. Neutralize the pH of the supernatant solution by adding 11 µL of 5.8 M HCl. Confirm a pH of ~7 by dipping litmus paper into each sample solution.
      CAUTION: Hydrochloric acid is a strong corrosive acid that can cause burns when in contact with skin (skin application or inhalation). Please wear gloves to prevent skin irritation while handling HCl.
   2. Add 90 µL of 100% trichloroacetic acid (TCA) to each tube and incubate tubes on ice for 30 minutes. The precipitation of proteins will turn the solution from clear to opaque. If this does not occur after 30 minutes, refrigerate the tubes for 1 hour. This is a potential stopping point, and samples can be stored at 4 °C overnight if needed.
      CAUTION: Trichloroacetic acid is corrosive. Please wear gloves when handling to prevent contact with skin and avoid inhalation.
   3. Centrifuge samples at 13,000 x g for 10 minutes at 4 °C. Carefully remove TCA supernatant by suctioning it out using a vacuum line attached to a fine glass micropipette tip (the same tip can be used across samples). Do not disturb the protein pellet by avoiding heavy suction and keeping an appropriate distance between pipette tip and the pellet.
   4. Wash pellet quickly with 100 µL of -20 °C acetone. This step removes any remaining TCA from the pellet, which can interfere with the subsequent Bradford assay; however, acetone will start to dissolve the protein pellet if left in contact too long, so acetone should be added then quickly extracted from the pellet within 5 seconds.
   5. Allow acetone to evaporate by placing tubes in a fume hood or refrigerator. Then, dissolve the protein pellet by adding 1 mL of 0.1 M NaOH to each tube. Fully dissolving the pellet may require several rounds of heating in a hot water bath, vortexing, and sonicating.
      NOTE: The longer the tubes sit in the fume hood or refrigerator and the drier the pellets get, the more difficult they will be to re-solubilize. It is suggested to dry the pellet for 30 minutes, and then check for the presence of acetone every 10-15 minutes until it is clear that the acetone has evaporated (no acetone fumes are detectable).
4. Mix standard solutions, dilute unknown sample solutions, and quantify the total protein content of unknown samples using the Bradford assay.
   1. Prepare bovine immunoglobulin G (IgG) standard solutions according to the concentrations listed in Table 1 (lyophilized or IgG stock solution can be mixed with deionized water to achieve each concentration). Store standards at 4 °C. In a 96-well plate, add 160 µL of each IgG standard solution in triplicate starting at the A1 position of the well plate (0 µg/µL in A1, A2, A3 position, 0.0125 µg/µL in B1, B2, B3 positions, etc.).
   2. Prepare a diluted NaOH solution by adding 50 µL of 0.1 M NaOH to 950 µL of distilled water. As a blank negative control, add 60 µL of this solution to the well plate in triplicate (G1, G2, G3 positions).
   3. Prepare dilutions of unknown samples by adding 50 µL of each sample solution to 950 µL of distilled water in a new 1.5 mL microcentrifuge tube. Then, add 60 µL of each diluted sample to the well plate in triplicate, starting at the H1 position. A 96-well plate should allow for the analysis of 6 standards, 1 blank, and 25 unknown samples when read in triplicate. Each well plate analyzed should contain its own IgG standard and blank samples.
   4. Add 100 µL of distilled water to all blank and unknown samples wells. Now each well should contain a total of 160 µL. Using a multi-channel pipette (8 channels), add 40 µL of Coomassie brilliant blue G-250 protein dye to every well in the first column of the well plate. Mix the dye with the samples by plunging several times. Repeat for all other columns, replacing pipette tips to avoid contamination.
      CAUTION: Coomassie brilliant blue G-250 is an irritant and contact with skin, eyes, or lungs should be avoided. Please wear gloves to prevent contact with skin. If contact with skin occurs, this product will stain.
   5. Use a needle to pop any bubbles present in the wells, as they may interfere with the microplate spectrophotometer reading. Clean the needle to avoid contamination between wells. Let the microplate incubate at room temperature for 5 minutes.
   6. Using a microplate spectrophotometer, record the absorbance values for each well at 595 nm.

Table 1. Standard curve calculations for the soluble protein assay. The amount of protein in each standard is calculated by taking the concentration of each standard and multiplying it by the amount of standard solution in each well (160 µL).