A rapid total protein extraction protocol for SDS-PAGE-based proteomic analysis of woody plants cultured

Our study developed rapid and simplified protein extraction protocol for SDS-PAGE-based proteomic analysis of in vitro poplar (Populus x berolinensis K. Koch.) leaves. Different approaches to tissue fixation (fresh, liquid nitrogen, -80°C) and homogenization (manual grinding, automated bead-beating) were evaluated, revealing consistent protein profiles across methods. The inclusion of protease inhibitors and β-mercaptoethanol during extraction had no significant effect, reflecting low protease activity in juvenile tissues and enabling cost and labor savings. Protein extraction protocols with and without an acetone precipitation step were compared. While precipitation improved resolution for specific protein fractions, some protein bands were better visualized without precipitation, suggesting partial protein loss or masking during precipitation. Both ProteOrange and Coomassie Brilliant Blue stains detected proteins effectively, with ProteOrange offering enhanced contrast. The findings support using a simplified, non-precipitation protocol combined with bead-beating homogenization and omission of protease inhibitors as an efficient, cost-effective method for proteomic analysis of in vitro woody plants. This protocol facilitates rapid, reproducible protein extraction, aiding physiological assessment and molecular research in woody plant biotechnology.