Protein is the executor of physiological functions and the direct embodiment of life phenomena. The study of protein structure and function will directly elucidate the change mechanism of life under physiological or pathological conditions. The existence form and activity patterns of proteins themselves, such as post-translational modifications, protein-protein interactions, and protein conformation, still rely on direct research on proteins to solve.
Principle
Proteins can be precipitated by certain organic solutions. On the one hand, the addition of organic solvents such as methanol, ethanol, and acetone to water reduces the volume dielectric constant and increases the attraction of opposite charges. On the other hand, these organic solvents are strongly hydrophilic reagents that compete for hydration water on the surface of protein molecules, destroying the hydration layer on the surface of protein colloid molecules and causing the molecules to aggregate and precipitate. The precipitation effect is better at the isoelectric point. Organic solvents can denature proteins at room temperature and slow down the deformation speed at low temperatures. Therefore, protein precipitation with organic solvents should be carried out under low temperature conditions. For example, when using acetone to precipitate proteins, it must be carried out at a low temperature of 0 to 4°C. After proteins are precipitated with acetone, they should be separated immediately, otherwise the proteins will be denatured. Trichloroacetic acid (TCA) precipitates proteins mainly based on the following aspects: TCA can form insoluble salts with proteins under acidic conditions; As a protein denaturant, TCA changes the protein conformation, exposing more hydrophobic groups, causing them to aggregate and precipitate.
Reagent Preparation
- Extraction buffer A: acetone solution containing 10% trichloroacetic acid.
- Extraction buffer B: 0.07% β-thioethanol in acetone.
Procedures
- Pre-cool the prepared extraction buffers A and B at -20°C.
- Take 2 g of tomato fruit and put it into a pre-cooled mortar, add liquid nitrogen, and grind it thoroughly to powder (add a little PVPP).
- Add 3 times the volume of extraction buffer A to the 1.5 mL tube, add the powder into the tube, mix well and store overnight at -20°C.
- Place the tube into a refrigerated centrifuge, adjust the temperature to 4°C, centrifuge at 40,000 g for 1 h, and discard the supernatant.
- Resuspend the pellet in an equal volume of pre-cooled extraction buffer B, place the tube in a refrigerated centrifuge, adjust the temperature to 4°C, and centrifuge at 40,000 g for 1 h (repeat once), and discard the supernatant.
- Dry the precipitate in air at room temperature.
- Fully dissolve the precipitate with the minimum volume of lysis solution, and vortex to aid dissolution.
- Place the tube into a refrigerated centrifuge, adjust the temperature to 15°C, and centrifuge at 40,000 g for 1 h. The supernatant is the obtained protein sample. It can be divided into parts and stored at -80°C for later use. Temporary storage can be placed at 4°C.
Note:
- Trichloroacetic acid (TCA) is highly corrosive, so be careful when using it.
- The last step of extraction (step 8) is centrifuged to ensure there is no precipitation, which may be necessary multiple times. It may take longer each time.
- During the protein extraction process, attention needs to be paid to the protection of proteins. Some proteins are easily degraded. Protease inhibitors, reducing agents, etc. can be added, and operations should be kept at low temperatures as much as possible.
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