Fluorescence Recovery after Photobleaching (FRAP) Assay to Measure the Dynamics of Fluorescence Tagged Proteins in Endoplasmic Reticulum Membranes of Plant Cells

• Published in: Bio-protocol Vol. 4; no. 20
• Main Authors: José Navarro, Marta Serra-Soriano, Vicente Pallás
• Format: Journal Article
• Language: English
• Published: Bio-protocol LLC 01-10-2014
• ISSN: 2331-8325
• DOI: 10.21769/BioProtoc.1268

In this protocol, we used fluorescence recovery after photobleaching (FRAP) to measure the influence that some mutations and drug treatment have on mobility of a green fluorescent protein (GFP)-fused viral transmembrane protein into endoplasmic reticulum membranes (Serra-Soriano et al., 2014). The proteins of interest were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermic cells by agro-infiltration. To minimize transient overexpression artifacts, fluorescence intensity values were gathered at 36 hpi using an inverted Zeiss LSM 780 confocal microscope. Only epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins were used for further experiments. To examine the role of actin polymerization in the mobilization of GFP-tagged proteins, we pretreated tissue samples either with latrunculin B, an inhibitor of actin polymerization, or with DMSO as control. The generated fluorescence recovery curves were used to obtain the percentage of maximum fluorescence recovery (MFR), which corresponds to the mobile fraction, and the half-time of maximum recovery (t1/2) values.