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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling

Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education) and Biotechnology Research Center, Southwest Univ., Chongqing 400716, China

^* Corresponding author (yangxy94{at}swu.edu.cn).

An efficient protein extraction method to ensure successful separation by two-dimensional electrophoresis (2-DE) is a critical step for any proteomics study. The objective of this study was to compare four previously published protein extraction protocols to identify a suitable protein extraction method to extract total proteins from cotton (Gossypium hirsutum L.) seedlings, a recalcitrant plant tissue. The results suggested that trichloroacetic acid/acetone precipitation combined with phenol extraction method (Method D) produced the purest sample and the most proteins spots (321), particularly basic proteins in the 2-DE image. The extraction method combining acetone precipitation and phenol (Method B) gave 216 spots in the 2-DE image. The trichloroacetic acid/acetone precipitation method (Method A) is not suitable for the cotton seedlings because only small molecular weight proteins were visualized in the 2-DE image. The phenol extraction method (Method C) showed 240 spots in the 2-DE gel but gave higher gel background. We subsequently selected and optimized Method D to extract protein from cotton seedlings. After optimization, more than 900 spots were detected on the 2-DE gel with pH 3–10 nonlinear gradient strip and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel using Coomassie brilliant blue G-250 staining. Our results suggest that the optimized Method D is expected to have excellent applications in proteomic studies of cotton seedlings.

Abbreviations: 2-DE, two-dimensional gel electrophoresis • 2-ME, β-mercaptoethanol • CHAPS, 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid • DTT, dithiothreitol • EDTA, ethylene diamine tetraacetic acid • IEF, isoelectric focusing • IPG, immobilized pH gradient • Mr, molecular mass • PEG, polyethylene glycol • pI, isoelectric point • PMSF, phenylmethanesulfonyl fluoride • PVPP, polyvinylpolypyrrolidone • SDS, sodium dodecyl sulfate • SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis • rubisco, ribulose bisphosphate carboxylase/oxygenase • TCA, trichloroacetic acid