Alfalfa stem cell wall protein extraction experiment
The plant cell wall is a highly dynamic and chemically active component of the plant cell. The main components of the cell wall are polysaccharides, as well as proteins that make up about 10% of the cell wall volume. These proteins are difficult to isolate with high purity from the carbohydrate complex matrix. The matrix not only sequesters proteins but is also a contaminant for subsequent 2-DE analysis. This experiment is based on the "Guide to Plant Proteomics Experiments" [France], edited by H. Thielemant, M. Zivi, C. Damerweiler, and V. Mitschine.
Operation method
Alfalfa stem cell wall protein extraction
Materials and Instruments
Sodium acetate NaCl Ascorbic acid Homogenization buffer Elution buffer Move 3.1 Tissue fragmentation, cleaning, and gel testing for purity For more product details, please visit Aladdin Scientific website.
Liquid nitrogen Mortar and pestle Brinell's funnel Vacuum chamber or pump
( 1 ) Collect 7-8 g of mature (internodes 4-6) alfalfa stems. This weight of mature stem tissue should contain more than 1 mg of cell wall proteins in this quantity ( see Note 2). Store at -80°C after collection or proceed immediately to the next step.
( 2 ) Place all solutions on ice.
( 3 ) Cut stems into small pieces, 1 to 2 cm in length, to make grinding easier.
( 4 ) Thaw frozen tissue (see Note 3).
( 5 ) Grind with mortar and pestle until the tissue foams (see Note 4). Make sure the tissue is ready for grinding. Grind without buffer, then add 10 ml of homogenization buffer and grind again.
( 6 ) Place the ground tissue on a nylon mesh filter paper lined with a Brinell funnel and vacuum filter. Spread the tissue evenly on the filter paper with a spatula. Rinse the mortar and pestle with 15 ml of homogenization buffer and use it to wash/filter the remaining cell debris (see Note 5 ). Rinse the debris particles more than 3 times each time with 25 ml of buffer, for a total of 100 ml (approx. 14 ml buffer/g) per 7-8 g of initial tissue (see Note 6). A vacuum pump is used to remove the washings. The eluate fraction is retained for purity determination (see Note 7). Appropriate methods for purity testing are SDS-PAGE analysis (Fig. 10-1A, lane 1) or Western hybridization with antibodies to known intracellular proteins (Fig. 10-1B, band 2).
( 7 ) Wash the debris with 50 ml of wash buffer 1 (sodium chloride wash, see Note 8). Pour it over the cell debris placed on a nylon filter membrane and remove it by vacuum filtration. Turn off the vacuum pump when adding the wash solution and make sure that the wash solution touches all the debris. Retain the wash fraction to determine purity (Figure 10-1A, lane 2; Figure 10-1A, lane 3).
( 8 ) Repeat the wash twice with 50 ml of distilled water (Figure 10-1A, lane 3; Figure 10-1B, lane 4).
( 9 ) Rinse 5 times with 50 ml of ice-cold acetone (see Note 9; Figure 10-1A, lane 4).
( 10 ) Repeat step 9 (Figure 10-1A, lane 5).
( 11 ) Finish with a final wash with 50 ml of wash buffer 2 (10 mmol/L sodium acetate; Figure 10-1A, lane 6; Figure 10-1B, band 5). 
3.2 Extraction of cell wall proteins
(1) Place the cell debris into a 50 ml tube on ice and add 7.5 ml of Extraction Buffer 1 (calcium chloride buffer). Place the tube to one side to increase the surface area of the cell debris in contact with the extraction buffer. Fix on a shaker and shake gently for 30-45 min.
( 2 ) Collect the protein extract (see Note 10) and repeat the extraction for 30-45 min by adding the appropriate amount of Extraction Buffer to the water (see Note 11).
( 3 ) Remove the second protein extract and combine the resulting extract with the first extract. Add 15 ml of Extraction Buffer 2 (lithium chloride buffer) and leave the extract on ice for at least 45 min or overnight in a cryostat (see Note 12 and Note 13).
( 4 ) Centrifuge the extracts slightly at 4°C (~1000 g) to remove the particulate matter before placing them in the concentrator.
3.3 Protein concentration and desalting
( 1 ) Concentrate the sample in the centrifuge concentrator according to the instructions in the instrument manual (see Note 14). We prefer to use a 15 ml Amicon ultracentrifuge filtration device. Extracts can be separated or combined at this step. Concentrate to 100 to 150 μl.
( 2 ) The ReadyPrep 2-D Removal Kit from Bio-Rad accommodates salt concentrations up to 1 mol/L. Since the lithium chloride extract is in 3 mol/L lithium chloride, desalting is accomplished by adding 2 volumetric volumes of distilled water to the concentrated sample, which is then concentrated again. Transfer the protein solution into a microfuge tube, and a 200 μl tip minus the tip may help to transfer the sample from the concentrator. Flush the membrane of the concentrator with a small volume (~100 μl) of distilled water or buffer and combine with the concentrated protein. 
3.4 Protein Wash, Sample Hydration and Concentration Determination
( 1 ) Determine the protein concentration, using the Bradford method or equivalent.
( 2 ) Cell wall preparations at this stage can be used for SDS-PAGE without any further washing (Figure 10-2, lanes 1 and 2), but the protein bands will not be as clear as those that have been treated with commercial kits and solubilized with SDS sample buffer (Figure 10-2, lanes 3 and 4).
( 3 ) For 2-DE (Fig. 10-3A, calcium chloride and lithium chloride extracts combined; Fig. 10-3B, calcium chloride extracts; Fig. 10-3C, lithium chloride extracts), treatment is performed with the ReadyPreP Cleanup Kit (see Note 15) or other commercially available kits (see Note 16). The Bio-Rad kit works at 1 to 500 μg of protein in 100 μl. 