Analysis of Cell Wall Proteins during Xylem Vessel Secondary Cell Wall Formation in Cell Culture
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Proteins constitute to about 10% of the cell wall mass; nevertheless they are essential for maintaining the physical and biological functions in a plant cell. Yet, unidentified functional proteins ...
Proteins constitute to about 10% of the cell wall mass; nevertheless they are essential for maintaining the physical and biological functions in a plant cell. Yet, unidentified functional proteins might still exist in the cell wall. The completion of Arabidopsis genome has allowed the identification of cell wall proteins by using mass spectrometry (MS) techniques. However, it should be noted that several constraints arises during the extraction of cell wall proteins (i) proteins may be embedded in the polysaccharide matrix of cellulose, hemi-cellulose and pectin (ii) some proteins are difficult to solubilise (iii) some proteins undergo post-translational modifications and (iv) lack of surrounding membrane may result in a loss of cell wall proteins. So, specific extraction procedure should be used. Our strategies involved cell wall preparation through mechanical grinding (ball miller, mortar and pestle, sonication) followed by purification with increasing concentration of sucrose and sequential extraction using different concentration of salts. In addition, SDS-PAGE followed by western blotting was done to check the purity of cell wall prepared. Finally, proteins from the cell wall fractions (resultant CW5-pellet and 0.1M CaCl2 extraction) were identified using MS analysis and Arabidopsis thaliana database search. Result: During the cell wall preparation, we observed that mechanical disruption of Arabidopsis cell was the most efficient with Freezer Mill method. In consistent to this, we purified the cell sample homogenized through this method. Upon SDS-PAGE and western blotting using anti-tubulin antibody as the primary antibody, we observed a 55kDa tubulin band only in the first washing point of both basal and induced sample. This implied that the purification strategy that we had adopted was efficient. Furthermore, the resultant CW5 pellet and 0.1M CaCl2 extraction were subjected for proteomic analysis. It revealed that 44.3% of the identified proteins were cell wall proteins in the resultant CW5-pellet (induced) compared to 39.3% in the basal sample. It was also found that some of the cell wall proteins were released during 0.1M CaCl2 extraction. Conclusion: This method of preparing cell wall through mechanical disruption, fractionation through increasing density cushions and extraction of proteins with different concentration of salts provides a good cell wall preparation technique. In fact, the principle of this technique can offer a stage for studying cell wall proteome.
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