Protein is the material basis of life, the basic organic matter that constitutes cells, and the main carrier of life activities. Without protein there is no life. Therefore, it is a substance closely related to life and various forms of life activities. Every cell and every important component in the body has proteins involved.
Principle
Protein sample preparation mainly includes: breaking and lysing tissue cells, releasing proteins from tissue cells, and removing non-protein components such as nucleic acids and lipids. Detergents such as Trition X-100 and NP40 can release proteins from cells by dissolving cell membranes. Various proteolytic enzymes ubiquitous in cells can cause protein decomposition, so low-temperature operation and the addition of some corresponding protease inhibitors (such as diisopropylfluorophosphate, p-chloromercuric acid, phenylmethylsulfonyl fluoride, etc.) and metal chelators (EDTA, etc.) are beneficial to protecting the integrity of the target protein.
Procedures
- Weigh 0.5 g of wheat leaves, cut into pieces, and place in a mortar pre-cooled at -20°C.
- Add 2.5 mL Tris-HCI extraction buffer and grind on ice to form a homogenate.
- Use a 1 mL scissored pipette tip to draw the homogenate from the mortar into a 1.5 mL centrifuge tube on ice. Centrifuge at 5,000 r/min for 10 min at 4°C (if the speed is higher and the time is longer, the protein content obtained will be different).
- Pipette 200 µL of the supernatant into a 1.5 mL centrifuge tube, add 1 mL of acetone (containing 10 mmol/L disulfide ethanol), mix well, and precipitate at -20°C for 1 h.
- Centrifuge the precipitate at 10,000 r/min for 10 min and discard the supernatant. The precipitate was washed twice with 80% acetone (centrifuged at 10,000 r/min for 3 minutes each time), dried at room temperature for 5-10 min, and stored at -20°C for later use.
- Take another 1-20 µL sample from the supernatant in step 3, add it to 2 mL of 30 mmol/L Tris-HCI buffer, and place it in a cuvette. Measure OD250 and OD280 and record the data (if the cuvette has a capacity of 2 mL, the sample amount depends on the concentration so that the OD is between 0 and 1).
- Calculate the protein concentration according to the formula:
When OD280/OD260<1.5, use the Lowry-Kalokar formula:
Protein content of the solution in the cuvette (mg/mL)=1.45 OD280～0.74OD260
When OD280/OD260>1.5, use Lambert-Beer's law:
Protein content of the solution in the cuvette (mg/mL)=OD280/KL=(OD280/6.3×1)×10 g/L
In the formula: K is the molar extinction coefficient.
Note:
- All processes of total plant protein extraction require preserving the integrity of the biological macromolecular structure during operation. Preserve the activity of plant proteins and prevent protein denaturation and degradation. Since the spatial structure mainly relies on the existence of hydrogen bonds, salt bonds and van der Waals forces, exposure to acids, alkalis, high temperatures, severe mechanical effects and strong radiation can lead to loss of activity. Therefore, the conditions chosen must be very mild. At the same time, attention should be paid to preventing contamination of heavy metal ions, cells' own enzymes and other toxic substances in the system.
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