Journal Article

Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts

Biswas, Subhankar, Srivastava, Vaibhav, Singh, Sushil Kumar, Kumar, Sanjiv, Srivastava, Akanksha, Chaurasia, Neha, Yadav, Sandhya, Mishra, Yogesh

2020

Overview

Objective Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results We compared three different protein extraction methods, i.e.,1.5 M Tris–HCl (pH 8.8), 50 mM Tris–HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)–acetone, in a liverwort Dumortiera hirsuta . Among these methods, 50 mM Tris–HCl (pH 7.5) extraction, followed by 20% TCA–acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum . Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acetone / Analysis / Biomedical and Life Sciences / Biomedicine / Chemical precipitation / Chemical properties / Dumortiera hirsuta / Electrophoresis / Ethanol / Gel electrophoresis / Glycerol / Isoelectric focusing / Life Sciences / Liverworts / Marchantia paleacea / Marchantiophyta / Mass spectrometry / Mass spectroscopy / Medicine/Public Health / Metabolites / Methods / Optimization / pH effects / Plagiochasma appendiculatum / Plant proteins / Polyvinylpolypyrrolidone / Proteins / Proteomics / Rehydration / Research Note / Scientific imaging / Solubilization / Spectroscopy / Studies / Two-dimensional gel electrophoresis (2-DE) / Water