Cereal plant seed protein extraction experiment
Cereal plant seed protein extraction experiment
Seeds may contain different components, like starch and polysaccharides, which can severely reduce the amount of protein extracted. The proteins in cereal plant seeds are usually divided into four groups according to their solubility: albumin, globulin, prolamin and glutelin, which can be extracted separately. This experiment is based on the "Guide to Plant Proteomics Experiments" (France), H. Tillmant, M. Zivi, C. Damerwell, V. Mitschine, eds.
Operation method
Cereal plant seed protein extraction
Materials and Instruments
Albumin Globulin Move 3.1 Universal methods for total seed protein extraction For more product details, please visit Aladdin Scientific website.
Extraction solution Rinsing solution Dissolving solution Burning agent Glycerin solution
The purpose of using the universal method is to extract total protein from milled seeds, regardless of the specific protein species. This method was proposed by Damerval et al [ 9] and Granier [ 10] and it can be used for all seeds, see Chapter 1.
This method is particularly suitable for the extraction of proteins not only from green ripe seeds, but also from cereal grains at an early stage of formation, the seed coat, the embryo or the developing endosperm. Some special tissues such as the pasty layer, the embryo and the endosperm can be separated from developing as well as from mature seeds. Unlike isolation of the embryo, which is easy, isolation of the pasty layer requires time and experience, and many seeds have to be dissected under the microscope to obtain enough material for protein extraction and bidirectional electrophoresis.
There is variation in the seeds harvested from a single plant, so it is important to collect immature seeds from representative spikes, inflorescences, pods, and so on.
Depending on the method used, i.e. precipitation prior to lysis (see below) or direct lysis (see below), the amount of protein extracted or the protein profile on the bidirectional electrophoresis gel will vary. Regardless, once a method has been applied, the entire experiment must follow the operational procedures of that method.
1. Precipitation prior to solubilization
Green ripe material from spikes, inflorescences, pods, etc. was weighed immediately after collection. These green ripe seed materials were frozen before grinding.
( 1 ) The ground powder (100 mg) is immediately added to 1 ml of pre-cooled TCA/acetone extraction buffer (see Notes 3-5). After overnight precipitation, centrifuge at 15000 g at 4°C for 15 min.
( 2 ) The precipitate is washed twice with rinsing solution, each time followed by centrifugation at 15000 g for 15 min at 4°C. The supernatant is discarded (see Note 6).
( 3 ) The precipitate obtained from 100 mg of material is air-dried to remove residual acetone and then stored at -80°C (see Note 7). This method allows the proteins to be highly concentrated before being solubilized by 1250 μl of solvent or by IEF buffers containing special solvents such as chaotrope and detergent.
2. Direct re-solubilization
Proteins in seeds can be extracted directly from the milled seeds without prior extraction and precipitation with TCA/acetone. Extraction of proteins directly from natural tissues with detergents and dissociatives is more efficient than extraction of proteins from precipitation, and this method prevents loss of proteins due to precipitation or incomplete resuspension. The methods described herein do not include precipitation using pre-cooled acetone, although pre-cooled acetone is useful in concentrating proteins and removing salts and other organic compounds. This acetone precipitation method was not proposed until recently [11] . Another recently reported method is the extraction of proteins from developing wheat kernels by enhanced solubilization with KCl [ 12, 13] . Proteins from barley were first presented using 5 mmol/L Tris-HCl, pH 7.5 and 1 mmol/L CaCl2 [14, 15]. Thiourea/urea lysis buffer containing various protease inhibitors, DNAase I, RNAase, Triton X-100, and DTT was used for Arabidopsis seeds. The method described here is mainly applicable to the extraction of endosperm proteins from cereal plants, which consist mainly of storage proteins (alkyd and glutenin). This method cannot exclude other proteins, especially since many albumin and globulin proteins are also extracted together with the storage proteins.
( 1 ) The material is weighed immediately after collection and then crushed (see Notes 8 and 9) or ground (see Note 10).
( 2 ) 500 μl of the dissolution solution is added to a centrifuge tube that already contains 40 mg of the material powder, vortexed, and incubated for 1.5 h at room temperature.
( 3 ) After ultrasonication (20 W, 20 s), the suspension was left for 30 min and centrifuged (9000 g, 5 min, 20°C). 250 μl of the supernatant was collected. Then 250 μl of supernatant was collected. The supernatant mainly contained storage protein.
( 4 ) Determine the protein concentration by using Caulmers Blue method or 2D Quant kit.
( 5 ) Add 20 μl of carrier ampholyte (selected according to the IPG strip gradient in the IEF procedure) and 4 μl of 4-vinylpyridine (see Note 11) to 250 μl of supernatant. Vortex for 15 min and add 254 μl of glycerol solution. Store at -80°C until use.
3.2 Extraction of specific proteins
1. Albumin and globulin
Simultaneous extraction of two proteins (water-soluble albumin and salt-soluble globulin) has been optimized for isoelectric focusing applications.
Therefore, the proteins obtained after protein extraction with sodium phosphate buffer [ 18 ] were first desalted, precipitated, and then resuspended in isoelectric focusing buffer. Recently, albumin and globulin from wheat seeds were successfully separated in bidirectional electrophoresis using this method [ 19 ]. Seeds (including seed coat, with or without embryo) are milled according to the method described in 3. 3.1 Section 2 or as described in Notes 8 to 10. Protein extraction is accomplished according to the following steps.
( 1 ) Add 100 mg of material powder to 1 ml of sodium phosphate buffer and stir continuously at 4°C for 2 h. Centrifuge ( 8000 g, 10 min, 4°C) and remove 500 μl of supernatant (see Note 13).
( 2 ) Dialyze with cold water at 4°C for 24 h according to the manufacturer's instructions (see Note 14).
( 3 ) The salt-removed sample solution is mixed with 1 ml of acetone, reacted and precipitated overnight at -20°C, then centrifuged (8000 g at 4°C for 5 min).
( 4 ) The supernatant is removed and the protein precipitate is rinsed three times with pre-cooled acetone, each time followed by centrifugation (8000 g at 4°C for 5 min). The precipitated proteins were air-dried and the residual acetone was removed.
( 5 ) Dissolve the precipitate in 250 μl of dissolution buffer, vortexing several times (see 3.2.1). After 1.5 h at room temperature, the solubilized proteins are sonicated (20 W, 20 s), the suspension is allowed to stand for 30 min, centrifuged (9000 g, 5 min, 20°C), and the supernatant is collected.
( 6 ) The supernatant containing the proteins was collected and supplemented with 20 μl of carrier ampholyte and 4 μl of 4-vinylpyridine. The solution was vortexed and allowed to stand for 15 min.
( 7 ) 254 μl of glycerol was added. The solution is stored at -80°C until analyzed by bidirectional electrophoresis ( see Note 12 ).
2. Amphoteric proteins
This protein consists of two forms of amino acid sequences, one sequence rich in hydrophilic amino acids (mostly Lys, Arg, and His) and the other sequence rich in hydrophobic amino acids (e.g., Val, Leu, and lie). Most membrane proteins are amphipathic. The extraction method described here is based on suitable modifications with other methods [ 20] to facilitate the extraction of amphoteric proteins from wheat seeds [ 21, 22].
( 1 ) The seeds were ground as described previously (see Notes 8-10).
( 2 ) 250 mg of powder was added to 7.5 ml of Triton X-114 solution, stirred at 4°C for 1 h, and centrifuged at 10,000 g at 4°C for 30 min.
( 3 ) The supernatant is heated at 37°C for 45 min for phase partitioning and then centrifuged at 5000 g for 10 min at 22°C. Carefully remove the upper light phase (see Note 15).
( 4 ) Add 5 volumes of precipitate (approximately 10 ml) to the amphiphilic protein-rich lower phase. Precipitate overnight at -20°C.
( 5 ) After centrifugation at 2000 g for 10 min at -8°C, the precipitate is rinsed three times with 10 ml of diethyl ether ethanol solution, each time followed by centrifugation at 2000 g for 10 min at -8°C. The precipitate is removed carefully (see Note 15).
( 6 ) Finally, the precipitate was rinsed with 5 ml diethyl ether, centrifuged and dried under vacuum at room temperature. The dried precipitate is stored at -80°C for later use. For isoelectric focusing, dissolve the dried precipitate in 250 μl of dissolution solution (see 3.2.1 ).
3. starch granule proteins
Starch is the major component of the endosperm of cereal plant seeds (70% to 75% of dry weight ). Starch grains originating from the starch-making body accumulate in the mature endosperm with seed formation. In cereal seeds, starch granules are distributed in different sizes ( Fig. 3-1 ). The method described below is used to extract proteins adsorbed on or present in starch grains in cereal plant seeds. This method was modified from studies on starch synthases in wheat starch [ 23,24] .
( 1 ) Crush 1 to 2 half-seeds with a mortar and pestle at room temperature and separate the ground tissue from the seed coat tissue (see Note 16).
( 2 ) Soak 40 to 60 mg of powder in 1 ml of deionized water at 4°C overnight. Centrifuge the suspension at 10000 g at 4°C for 2 min.
( 3 ) 300 μl of cold water is then added to the precipitate and vortexed to mix.
( 4 ) The suspension is layered with 1 ml of 90% (V/V) CsCl, allowed to stand for 5 min at 4°C, and then centrifuged at 14000 g for 5 min at 4°C. The supernatant is carefully removed.
( 5 ) 1 ml of rinse solution containing CHAPS and DTT is then added to the precipitate. Vortex to mix, heat at 37°C for 15 min, and vortex every 5 min. The suspension was centrifuged at 14,000 g, 4°C for 3 min and the supernatant was carefully removed.
( 6 ) The starch pellet precipitate is centrifuged (same parameters as before) and rinsed three times with water as follows: 1 ml of water is added to the precipitate, the suspension is vortexed and allowed to stand at room temperature for 5 min, then centrifuged at 14,000 g, 4°C, for 3 min. The supernatant is carefully removed each time.
( 7 ) Finally rinse with pre-cooled acetone for 5 min, vortex and centrifuge as in step (6). The starch pellet was dried, weighed, and stored at -20°C for later use.
( 8 ) Resuspend the starch pellet sediment with rinse solution at a ratio of 1 : 5 (m/V). Typically, 40 mg of dried starch precipitate can be vortex-suspended in 200 μl of freshly prepared solution containing 4% (m/V) CHAPS and 2% (m/V) DTT at room temperature (see Note 16). The suspension is allowed to stand for 5 min and heated at 100°C for 5 min to allow the starch granules to release their inclusions.
( 9 ) The suspension is cooled in ice for 10 min and centrifuged at 17000 g, 4°C for 15 min. The supernatant contains proteins strongly adsorbed on or present in the starch granules. The collected proteins can be stored at -80°C or used directly for bidirectional electrophoresis. For isoelectric focusing, 150 μl of dissolution solution (see 3.2.1 ) is added to 100 μl of supernatant.