title: 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.
authors.affiliation: Center of Excellence for Innovation in Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: Aphinya
authors.initials: A
authors.lastname: Wongpia
authors.forename: Sugunya
authors.initials: S
authors.lastname: Mahatheeranont
authors.forename: Khemika
authors.initials: K
authors.lastname: Lomthaisong
authors.forename: Hataichanoke
authors.initials: H
authors.lastname: Niamsup
mesh_terms: D000085:Acetates / Q000737:chemistry; D000096:Acetone / Q000737:chemistry; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D002021:Buffers; D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional; D012275:Oryza / Q000737:chemistry*; D019800:Phenol / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry; D036226:Seedlings / Q000737:chemistry*; D012639:Seeds / Q000737:chemistry*; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: D000085:Acetates; D002021:Buffers; D010940:Plant Proteins; D000096:Acetone; D019800:Phenol; D011092:Polyethylene Glycols; D014238:Trichloroacetic Acid; C018824:ammonium acetate