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

• Published in: Applied biochemistry and biotechnology Vol. 175; no. 2; pp. 1035 - 1051
• Main Authors: Wongpia, Aphinya, Mahatheeranont, Sugunya, Lomthaisong, Khemika, Niamsup, Hataichanoke
• Format: Journal Article
• Language: English
• Published: Boston Springer-Verlag 01.01.2015; Springer US; Springer Nature B.V
• Subjects: Acetates - chemistry; Acetone; Acetone - chemistry; Agricultural biotechnology; Analytic Sample Preparation Methods - methods; Aquatic plants; Biochemistry; Biotechnology; Buffers; Chemical Precipitation; Chemistry; Chemistry and Materials Science; crop yield; Electrophoresis; Electrophoresis, Gel, Two-Dimensional; Fractionation; molecular weight; Oryza - chemistry; Oryza sativa; phenol; Phenol - chemistry; Phenols; Plant Proteins - analysis; Plant Proteins - chemistry; Plant Proteins - isolation & purification; polyethylene glycol; Polyethylene Glycols - chemistry; proteins; Proteomics; Rice; Sample preparation; Seedlings; Seedlings - chemistry; Seeds; Seeds - chemistry; trichloroacetic acid; Trichloroacetic Acid - chemistry; two-dimensional gel electrophoresis; Rice seedling; Rice seed; Sample preparation; Proteomics; Two-dimensional gel electrophoresis
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1333-0

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.