Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling

Abstract

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.