Evaluation of sample preparation methods from rice seeds and seedlings suitable for two-dimensional gel electrophoresis.

Abstract

In a proteomic study, sample preparation is very important because it affects the quality of protein profiles on two-dimensional gel electrophoresis (2-DE). This study investigated the suitability of four protein extraction methods-direct lysis buffer extraction, trichloroacetic acid (TCA)/acetone precipitation, phenol extraction, and polyethylene glycol (PEG) fractionation-from rice seeds and seedlings (Oryza sativa L. ssp. indica cv. Khao Dawk Mali 105). The effectiveness of these methods was evaluated by the protein quantity and the 2-DE profiling quality. This included the number of protein spots, the consistency and uniqueness of protein spots, and their distribution in different ranges of pI and molecular weight (M r ). For protein quantity, the phenol and direct lysis extraction methods gave the highest protein yield in rice seeds and rice seedlings, respectively. However, in terms of the quality of 2-DE profiles, samples prepared by the TCA/acetone and phenol methods exhibited higher protein resolution and more spots than the protein profile derived from direct lysis extract. In addition, TCA/acetone method had the efficiency for high M r protein detection. PEG fractionation provided the best 2-DE pattern in terms of resolution, number of spots, minimal streaking, and reproducibility. Moreover, PEG fractionation was better for determining low M r basic proteins.