Establishment of the Optimal Conditions for Two-Dimensional Gel Electrophoresis of Papaya Seed Proteome

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Title:	Establishment of the Optimal Conditions for Two-Dimensional Gel Electrophoresis of Papaya Seed Proteome
Authors:	Roan, SF (Roan, Su-Feng) Hsiung, TC (Hsiung, Tung-Chuan) Yang, KY (Yang, Kai-Yun) Liu, WT (Liu, Wei-Ting) Chang, IF (Chang, Ing-Feng) Wu, CT (Wu, Chun-Ta) Wakana, A (Wakana, Akira)
Contributors:	Dept Hort & Biotechnol
Keywords:	papaya seed protein extraction proteomics two dimensional gel electrophoresis zinc-reverse stain
Date:	2013-09
Issue Date:	2014-02-24 16:15:49 (UTC+8)
Abstract:	Papaya seeds are considered as a recalcitrant material for protein preparation in two dimensional gel electrophoresis (2 DE) because of abundance of interfering compounds. We examined two protein extraction methods, the classical trichloroacetic acid (TCA) / acetone precipitation (TCA A) and phenol ammonium acetate methanol precipitation (P AA M) methods, to evaluate their compatibility and efficiency of extracting papaya seed proteins in SDS-PAGE and two dimensional (2D) gel electrophoresis analysis. Compared with TCA A method, P AA M method showed better results, i.e., it increased at least five distinguished bands in SDS-PAGE, reduced problems of streaking and smearing in 2D gel, and enhanced resolution of detectable spots from 124 +/- 27 to 717 +/- 32. Comparison between traditional Coomassie Brilliant Blue R-250 staining and zinc reverse staining was also conducted. The result showed that seven times more protein spots could be identified from 2D gel stained with zinc reverse stain procedure. The resolution of 2D gel analysis for papaya seed proteome was further improved by utilizing pH 4-7 isoelectric focusing gel instead of pH 3-10 one. The protein spots picked from 2D gel through the protocol that we established were compatible with identification assessments of LC-MS/MS. Our data will serve as a platform and reference for future research in proteomics of papaya seeds or other recalcitrant plant materials.
Relation:	JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY Volume: 58 Issue: 2 Pages: 239-246
Appears in Collections:	[Department of Horticulture] journal articles

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