Soluble proteins refer to proteins that can be dissolved in water or other solvents in the form of small molecules. Most of the soluble proteins in plants are enzymes involved in various metabolisms and are one of the important indicators of plant stress resistance.
Principle
In this experiment, plant soluble protein extraction buffer was used to extract soluble proteins from plant samples that were quickly frozen and crushed in liquid nitrogen. The above-extracted plant protein is concentrated and simply purified using the precipitation method with water-soluble organic solvents (acetone, methanol, ethanol, etc.). The protein precipitate is washed with acetone and 80% acetone solution and then dissolved in loading buffer. The resulting protein solution can be directly used for polyacrylamide gel electrophoresis analysis.
Reagent Preparation
- Soluble protein extraction buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% β-mercaptoethanol, 1 mmol/L PMSF): Use a 5 mL micropipette to add 1.56 mL of 1 mol/L Tris-HCl (pH6.8) into a clean 50 mL beaker. Add 2.5 mL glycerol and 4.4 mg PMSF, then add approximately 15 mL double-distilled water, mix well, and adjust the volume to 24.5 mL. Place in a 4°C refrigerator until use (add 1/50 times the volume of β-mercaptoethanol before use).
- Loading buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% β-mercaptoethanol, 2% SDS): Use a 5 mL pipette to add 1.56 mL of 1 mmol/L Tris-HCl (pH 6.8) into a 50 mL beaker. Add 2.5 mL of glycerol and 5 mL of 10% SDS, then add 10 mL of double-distilled water and mix evenly. Adjust the volume to 24.5 mL and place it in a 4°C refrigerator for later use (add 1/50 times the volume of β-mercaptoethanol before use).
Procedures
- Put 3 mL of soluble protein extraction buffer into a 15 mL centrifuge tube and pre-cool on ice.
- Take 0.2 to 0.5 g of Arabidopsis thaliana leaves, put them into a mortar, freeze them quickly in liquid nitrogen, and then grind them to powder.
- Transfer the crushed leaves to the above-mentioned centrifuge tube containing 3 mL of soluble protein extraction buffer, shake and mix well, and place on ice for at least 1 hour. During this period, keep shaking the centrifuge tube to allow the extraction buffer to fully interact with the plant material.
- Centrifuge at 18000 r/min and 4°C for 30 min. Transfer the supernatant to another 15 mL centrifuge tube and discard the pellet.
- Slowly add 4 times the volume of 100% acetone to the supernatant, shake well, and place it in a -18°C refrigerator for 1 hour.
- Centrifuge at 18000 r/min for 30 min at 4°C. Discard the supernatant, add 3 mL of 100% acetone into the centrifuge tube, mix well with a pipette tip, and place in a -18°C refrigerator for 1 hour. Repeat once.
- Centrifuge at 18000 r/min for 30 min at 4°C and discard the acetone. Place in a -20°C refrigerator to air dry for 20 minutes.
- Add 250 µL loading buffer, mix well, and place on ice for 30 minutes.
- Centrifuge at 18000 r/min and 4°C for 30 min. Transfer the supernatant to a 1.5 mL centrifuge tube and freeze it at -70°C.
Note:
- Grind thoroughly and grind the plant material until it turns white.
- During the extraction process with protein extraction buffer, the plant material should be continuously shaken to fully interact with the extraction buffer.
- Add 100% acetone and shake the precipitate vigorously to allow electrophoresis interference substances such as chlorophyll to remain in the acetone phase.
- If the protein precipitate after 100% acetone cleaning contains green components, the number of acetone cleaning times can be increased to make the precipitate appear white.
- The acetone in the precipitation must be evaporated completely, otherwise it will affect the dissolution of the protein. Freeze vacuum drying can be used to remove residual acetone in the precipitate.
- The volume of loading buffer added should be appropriate, and too much should not be added to affect the concentration of the final protein solution. Adding too little will prevent the protein from fully dissolving.
- After adding the loading buffer, stir the precipitate evenly with a pipette tip to fully dissolve the precipitate.
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