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Author:
Date:
2014-10-20
[Abstract] 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 ...
[摘要] 在该协议中，我们使用光漂白（FRAP）后的荧光恢复来测量一些突变和药物治疗对于将绿色荧光蛋白（GFP） - 融合的病毒跨膜蛋白移动到内质网膜中的影响（Serra-Soriano， et al。，2014）。感兴趣的蛋白质通过农杆菌浸润在烟草（Nicotiana benthamiana）（本塞姆氏烟草）表皮细胞中瞬时表达。为了使瞬时过表达伪像最小化，使用倒置Zeiss LSM 780共聚焦显微镜在36hpi收集荧光强度值。只有表现出中等表达水平和通过GFP标记的蛋白的ER的均匀分布的表皮细胞用于进一步的实验。为了检查肌动蛋白聚合在GFP标记的蛋白质的动员中的作用，我们用拉特管素B，肌动蛋白聚合的抑制剂或用DMSO作为对照预处理组织样品。使用所产生的荧光恢复曲线来获得对应于流动级分的最大荧光回收率（MFR）的百分比和最大回收的半衰期（t 1/2）/sub>）值。
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