engleski

Optimisation of 2D-PAGE protocols for proteomic analysis of recalcitrant plant tissues

Plant tissues contain relatively low amounts of proteins whose extraction is often difficult due to presence of other compounds such as rigid cellulosic cell wall, storage polysaccharides, lipids and contaminating compounds that can result in protein degradation or modification. It is therefore important to optimize protein extraction and to establish a robust protocol for two dimensional electrophoresis (2DE). Here, we have evaluated acetone, trichloroacetic acid-acetone and phenol extraction protocols on recalcitrant plant tissues: Beta vulgaris L. cell line, Mammillaria gracilis Pfeiff. in vitro plants and Sempervivum tectorum L. leaves. Protein yield was determined by measuring protein concentration of the extracts with a modified Bradford assay. The spectrum of normalized protein extracts in the range from 180 to 900 nm was performed to determine the extent of left-over contamination, followed by a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins. Although phenol extraction takes more time, it gave almost two-fold higher protein yield than other methods, and spectral analysis showed less contamination. SDS-PAGE showed that protein extraction using phenol is better than the other two methods, providing more distinct protein bands on the gels of both higher and lower protein size. These preliminary results led to conclusion that phenol method is highly suitable for protein extraction from recalcitrant plant tissues. Further comparison will include isoelectric focusing and 2DE of the proteins followed by statistic and bioinformatic analysis of visualized protein spots.

Beta vulgaris; Mammillaria gracilis; Sempervivum tectorum; electrophoresis; protocol

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