Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.
Methods in molecular biology (Clifton, N.J.)(2023)
摘要
Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.
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关键词
Actin,Cofilactin,Cofilin,Filopodia,Fixation,Growth cones,Immunolabeling,Permeabilization,Phalloidin,Rods,cryoEM,cryoET
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