Method for Extracting Plant Proteins Using a High-Speed Refrigerated Centrifuge
Release time:
2012-07-02
Overview:
Add an extraction solution three times the volume of the sample, and let it stand overnight at -20℃. Then, centrifuge (at 4℃ at 8000 rpm or higher for 1 hour) and discard the supernatant.
I. Methods for Extracting Plant Tissue Proteins
1. Based on the sample weight (add 3.5 ml of extraction solution per 1 g of sample; adjust the amount appropriately depending on the material), prepare the extraction solution and place it on ice.
2. Place the sample in a mortar and grind it using liquid nitrogen. After grinding, add the extraction solution and let it stand on ice for (3–4 hours).
3. Centrifuge at 8,000 rpm for 40 minutes at 4℃, or at 11,100 rpm for 20 minutes at 4℃.
4. Extract the supernatant—sample preparation is now complete.
Protein extraction solution: 300 ml
1. 1 M Tris-HCl (pH 8) 45 ml
2. Glycerol 75 ml
3. Polyvinylpyrrolidone (PVP) 6 g
This method is designed for SDS-PAGE, vertical slab gel electrophoresis!
II. Process for Extracting Plant Tissue Proteins
Chloroacetic Acid-Acetone Precipitation Method
1. Grind the leaves in liquid nitrogen.
2. Add an extraction solvent equal to three times the volume of the sample, and incubate overnight at -20℃. Then, centrifuge (at 4℃ at 8000 rpm or higher for 1 hour) and discard the supernatant.
3. Add an equal volume of acetone chilled in an ice bath (containing 0.07% β-mercaptoethanol), mix thoroughly, and then centrifuge at 4°C at 8,000 rpm or higher for 1 hour. Subsequently, vacuum-dry the precipitate and set it aside for later use.
4. Add lysis buffer before sample loading, and let it stand at room temperature for 30 minutes to ensure that the protein is fully dissolved in the lysis buffer. Then, centrifuge (at 15°C at 8,000 rpm or higher for 1 hour or longer—until no precipitate remains). The sample can be temporarily stored at 4°C for later use.
5. Quantify the protein using the Bradford assay, then aliquot it and store it at -80℃ for future use.
Reagent: Extraction solution: Acetone containing 10% TCA and 0.07% β-mercaptoethanol. Lysis buffer: Dissolve 2.7 g of urea and 0.2 g of CHAPS in 3 ml of sterile, deionized water (final volume: 5 ml); before use, add 65 μl/ml of 1 M DTT.
This method is particularly suited to two-dimensional electrophoresis, given its characteristics of few impurities and low ion concentration! Of course, it’s also applicable to one-dimensional electrophoresis—though in that case, the number of bands will be reduced.
III. Organization: Intestinal Mucosa
Objective: To detect the expression of apoptosis-related proteins by Western blot.
Steps for protein extraction using TRIPURE:
Add isopropanol to the protein-containing supernatant: (1.5 ml per 1 ml of TRIPURE used).
Invert and mix thoroughly, then let stand at room temperature for 10 minutes.
Centrifuge at 12,000 g for 10 min at 4°C; discard the supernatant.
Add 0.3 M guanidine hydrochloride/95% ethanol: (2 ml per 1 ml TRIPURE usage)
Shake, then let stand at room temperature for 20 minutes.
Centrifugation: 7500 g, 5 min, 4°C; discard the supernatant.
Repeat step 2 with 0.3 M guanidine hydrochloride/95% ethanol twice.
Add 2 ml of 100% ethanol to the precipitate.
Shake thoroughly to mix well, then let stand at room temperature for 20 minutes.
Centrifugation: 7500 g, 5 min, 4°C; discard the supernatant and dry the precipitate by blowing.
1% SDS dissolved precipitate
Centrifugation: 10,000 g, 10 min, 4°C
Take the supernatant and store it at -20°C (or it can be directly used for WESTERN BLOT).
Existing issue: After adding 1% SDS, the precipitate doesn't dissolve and remains a large chunk. After centrifugation at 4°C, an even greater amount of white precipitate appears—could it be SDS crystallization? When measuring the concentration, the actual content is only about 1 mg/mL.
Solution: Use a protein extraction kit. Additionally, the tissue should be of moderate size and thoroughly minced. Immediately add 2X buffer, then heat for 5–10 minutes—it yields excellent results.
4. Plant materials: rice seedlings, leaf sheaths, roots
1. Place a 200-milligram sample on ice and grind it finely.
2. Add lysis buffer, centrifuge at 10,000 rpm at 4°C for 5 minutes, and then collect the supernatant.
3. Repeat centrifugation for 5 minutes.
Lysis buffer: urea, NP-40, ampholine 2, MPVP-40
V. Preparation of Protein Samples
The extraction of protein samples from seedlings was performed according to the method described by Davermal et al. (1986).
After shredding 100 mg of the material, add 10 mg of PVP-40 (polyvinylpyrrolidone) and a small amount of quartz sand. Grind the mixture to a fine powder using liquid nitrogen. Then, add 1.5 ml of 10% trichloroacetic acid (prepared in acetone and containing 10 mM, or 0.07%, β-mercaptoethanol), mix thoroughly, and allow the mixture to precipitate at -20℃ for 1 hour. Next, centrifuge at 4℃ at 15,000 rpm for 15 minutes; discard the supernatant. Resuspend the precipitate in 1.5 ml of cold acetone (containing 10 mM β-mercaptoethanol), and again allow it to precipitate at -20℃ for 1 hour. Following the same centrifugation procedure as before, discard the supernatant. If necessary, further freeze-dry the precipitate obtained after resuspension in 80% acetone (containing 10 mM β-mercaptoethanol) under low temperature and vacuum conditions.
Add 20 μl (adjustable) of UKS solution [9.5 M urea, 5 mM potassium carbonate, 1.25% SDS, 0.5% DTT (dithiothreitol), 2% Ampholine (Amersham Pharmacia Biotech Inc., pH 3.5–10), 6% Triton X-100] per mg of dry powder. Incubate at 37°C for 30 min, stirring several times during the incubation period. Then, centrifuge at 16,000 r/min for 15 min at 28°C (note: at lower temperatures, a high concentration of urea may cause the solution to freeze). The longer the centrifugation time and the higher the centrifugal force, the better! The supernatant can then be used directly for electrophoresis. Alternatively, store the sample at -70°C.
6. Extraction of Proteins from Plant Roots
(1) Sample, liquid nitrogen grinding
(2) Tube for 1.5 ml centrifuge
(3) Add 700 µL of 1 M KH2PO4/K2HPO4
(4) 12,000 rpm, 4°C, 10–15 minutes
(5) Take the supernatant—the protein is contained within it.
1. Based on the sample weight (add 3.5 ml of extraction solution per 1 g of sample; adjust the amount appropriately depending on the material), prepare the extraction solution and place it on ice.
2. Place the sample in a mortar and grind it using liquid nitrogen. After grinding, add the extraction solution and let it stand on ice for (3–4 hours).
3. Centrifuge at 8,000 rpm for 40 minutes at 4℃, or at 11,100 rpm for 20 minutes at 4℃.
4. Extract the supernatant—sample preparation is now complete.
Protein extraction solution: 300 ml
1. 1 M Tris-HCl (pH 8) 45 ml
2. Glycerol 75 ml
3. Polyvinylpyrrolidone (PVP) 6 g
This method is designed for SDS-PAGE, vertical slab gel electrophoresis!
II. Process for Extracting Plant Tissue Proteins
Chloroacetic Acid-Acetone Precipitation Method
1. Grind the leaves in liquid nitrogen.
2. Add an extraction solvent equal to three times the volume of the sample, and incubate overnight at -20℃. Then, centrifuge (at 4℃ at 8000 rpm or higher for 1 hour) and discard the supernatant.
3. Add an equal volume of acetone chilled in an ice bath (containing 0.07% β-mercaptoethanol), mix thoroughly, and then centrifuge at 4°C at 8,000 rpm or higher for 1 hour. Subsequently, vacuum-dry the precipitate and set it aside for later use.
4. Add lysis buffer before sample loading, and let it stand at room temperature for 30 minutes to ensure that the protein is fully dissolved in the lysis buffer. Then, centrifuge (at 15°C at 8,000 rpm or higher for 1 hour or longer—until no precipitate remains). The sample can be temporarily stored at 4°C for later use.
5. Quantify the protein using the Bradford assay, then aliquot it and store it at -80℃ for future use.
Reagent: Extraction solution: Acetone containing 10% TCA and 0.07% β-mercaptoethanol. Lysis buffer: Dissolve 2.7 g of urea and 0.2 g of CHAPS in 3 ml of sterile, deionized water (final volume: 5 ml); before use, add 65 μl/ml of 1 M DTT.
This method is particularly suited to two-dimensional electrophoresis, given its characteristics of few impurities and low ion concentration! Of course, it’s also applicable to one-dimensional electrophoresis—though in that case, the number of bands will be reduced.
III. Organization: Intestinal Mucosa
Objective: To detect the expression of apoptosis-related proteins by Western blot.
Steps for protein extraction using TRIPURE:
Add isopropanol to the protein-containing supernatant: (1.5 ml per 1 ml of TRIPURE used).
Invert and mix thoroughly, then let stand at room temperature for 10 minutes.
Centrifuge at 12,000 g for 10 min at 4°C; discard the supernatant.
Add 0.3 M guanidine hydrochloride/95% ethanol: (2 ml per 1 ml TRIPURE usage)
Shake, then let stand at room temperature for 20 minutes.
Centrifugation: 7500 g, 5 min, 4°C; discard the supernatant.
Repeat step 2 with 0.3 M guanidine hydrochloride/95% ethanol twice.
Add 2 ml of 100% ethanol to the precipitate.
Shake thoroughly to mix well, then let stand at room temperature for 20 minutes.
Centrifugation: 7500 g, 5 min, 4°C; discard the supernatant and dry the precipitate by blowing.
1% SDS dissolved precipitate
Centrifugation: 10,000 g, 10 min, 4°C
Take the supernatant and store it at -20°C (or it can be directly used for WESTERN BLOT).
Existing issue: After adding 1% SDS, the precipitate doesn't dissolve and remains a large chunk. After centrifugation at 4°C, an even greater amount of white precipitate appears—could it be SDS crystallization? When measuring the concentration, the actual content is only about 1 mg/mL.
Solution: Use a protein extraction kit. Additionally, the tissue should be of moderate size and thoroughly minced. Immediately add 2X buffer, then heat for 5–10 minutes—it yields excellent results.
4. Plant materials: rice seedlings, leaf sheaths, roots
1. Place a 200-milligram sample on ice and grind it finely.
2. Add lysis buffer, centrifuge at 10,000 rpm at 4°C for 5 minutes, and then collect the supernatant.
3. Repeat centrifugation for 5 minutes.
Lysis buffer: urea, NP-40, ampholine 2, MPVP-40
V. Preparation of Protein Samples
The extraction of protein samples from seedlings was performed according to the method described by Davermal et al. (1986).
After shredding 100 mg of the material, add 10 mg of PVP-40 (polyvinylpyrrolidone) and a small amount of quartz sand. Grind the mixture to a fine powder using liquid nitrogen. Then, add 1.5 ml of 10% trichloroacetic acid (prepared in acetone and containing 10 mM, or 0.07%, β-mercaptoethanol), mix thoroughly, and allow the mixture to precipitate at -20℃ for 1 hour. Next, centrifuge at 4℃ at 15,000 rpm for 15 minutes; discard the supernatant. Resuspend the precipitate in 1.5 ml of cold acetone (containing 10 mM β-mercaptoethanol), and again allow it to precipitate at -20℃ for 1 hour. Following the same centrifugation procedure as before, discard the supernatant. If necessary, further freeze-dry the precipitate obtained after resuspension in 80% acetone (containing 10 mM β-mercaptoethanol) under low temperature and vacuum conditions.
Add 20 μl (adjustable) of UKS solution [9.5 M urea, 5 mM potassium carbonate, 1.25% SDS, 0.5% DTT (dithiothreitol), 2% Ampholine (Amersham Pharmacia Biotech Inc., pH 3.5–10), 6% Triton X-100] per mg of dry powder. Incubate at 37°C for 30 min, stirring several times during the incubation period. Then, centrifuge at 16,000 r/min for 15 min at 28°C (note: at lower temperatures, a high concentration of urea may cause the solution to freeze). The longer the centrifugation time and the higher the centrifugal force, the better! The supernatant can then be used directly for electrophoresis. Alternatively, store the sample at -70°C.
6. Extraction of Proteins from Plant Roots
(1) Sample, liquid nitrogen grinding
(2) Tube for 1.5 ml centrifuge
(3) Add 700 µL of 1 M KH2PO4/K2HPO4
(4) 12,000 rpm, 4°C, 10–15 minutes
(5) Take the supernatant—the protein is contained within it.
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