Azri, W., Ennajah, A. and Jing, M. 2013. Comparative study of six methods of protein extraction for two-dimensional gel electrophoresis of proteomic profiling in poplar stems. Can. J. Plant Sci. 93: 895-901. Protein extraction is a crucial step in two-dimensional gel electrophoresis (2-DE) analysis of proteins, since it can have significant impact on both the quantity and the quality of protein detection. The present study is a comparison between six previously published protocols of protein extraction (A, B, C, D, E, and F) aiming to determine a suitable method to extract total proteins from poplar stems, a recalcitrant plant tissue. The obtained results revealed that method F (optimized method B), combining detergents (CHAPS, Triton X-100, and low sodium dodecyl sulfate amounts) and chaotropes (thiourea and urea), gave the best solution for the problem of protein solubilization. Method F enabled the detection of more than 300 spots reproducible on the 2-DE gel with pH 4-7 immobilized pH gradient strips and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using silver staining. Our results suggest that Method F is expected to have excellent applications in proteomic studies of poplar stems.