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- W4210348527 abstract "Single-particle tracking (SPT) makes it possible to directly observe single protein diffusion dynamics in living cells over time. Thus, SPT has emerged as a powerful method to quantify the dynamics of nuclear proteins such as transcription factors (TFs). Here, we provide a protocol for conducting and analyzing SPT experiments with a focus on fast tracking (“fastSPT”) of TFs in mammalian cells. First, we explore how to engineer and prepare cells for SPT experiments. Next, we examine how to optimize SPT experiments by imaging at low densities to minimize tracking errors and by using stroboscopic excitation to minimize motion-blur. Next, we discuss how to convert raw SPT data into single-particle trajectories. Finally, we illustrate how to analyze these trajectories using the kinetic modeling package Spot-On. We discuss how to use Spot-On to fit histograms of displacements and extract useful information such as the fraction of TFs that are bound and freely diffusing, and their associated diffusion coefficients." @default.
- W4210348527 created "2022-02-08" @default.
- W4210348527 creator A5023851910 @default.
- W4210348527 creator A5049740611 @default.
- W4210348527 date "2022-01-01" @default.
- W4210348527 modified "2023-09-26" @default.
- W4210348527 title "A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis" @default.
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- W4210348527 doi "https://doi.org/10.1007/978-1-0716-2140-0_9" @default.
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- W4210348527 hasPublicationYear "2022" @default.
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