Isolation and Visualization of Plant Stress Granule-Associated Components via On-Beads Digestion and Co-localization Analysis
2024
Solis-Miranda, Jorge | Rubio-Ramos, Rafael | Gonzalez-Rodriguez, Santiago | Gutierrez-Beltran, Emilio | Ministerio de Ciencia e Innovación (España) | Junta de Andalucía | Solis-Miranda, Jorge [0000-0001-7140-2558] | Rubio-Ramos, Rafael [0000-0001-5074-0371] | Gonzalez-Rodriguez, Santiago [0009-0001-0405-2153] | Gutierrez-Beltran, Emilio [0000-0001-7978-3164] | Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
Chemicals and CAS Registry Numbers Arabidopsis Proteins
显示更多 [+] 显示较少 [-]Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.
显示更多 [+] 显示较少 [-]This work was supported by grants from the Ministerio de Ciencia e Innovacion, grant PID2020-119737GA-I00 (MCIN/AEI/10.13039/501100011033) and the Junta de Andalucia (ProyExcel_00587).
显示更多 [+] 显示较少 [-]Peer reviewed
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