Evaluation of Sample Preparation Methods from Rice Seeds and Seedlings Suitable for Two-Dimensional Gel Electrophoresis.

Wongpia, Aphinya; Mahatheeranont, Sugunya; Lomthaisong, Khemika; Niamsup, Hataichanoke

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 p I and molecular weight ( M). 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 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 basic proteins.

PROTEOMICS; GEL electrophoresis; EXTRACTION (Chemistry); ACETONE; POLYETHYLENE glycol

Applied Biochemistry & Biotechnology, 2015, Vol 175, Issue 2, p1035

0273-2289

Academic Journal

10.1007/s12010-014-1333-0