A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis
Name
20210327 MiMB Fast Single Particle Tracking Final.pdf
Description
Accepted version
Size
470.89 KB
Format
Adobe PDF
Checksum (MD5)
c8eeb23c57b3210dcdbcd3a5e889a370
Author(s)
Jha, Asmita
Hansen, Anders S
Date Issued
2022
Journal
Methods Mol Biol
Publisher
Springer US
Citation
Jha, Asmita and Hansen, Anders S. 2022. "A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis." Methods Mol Biol, 2458.
Version
Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1007/978-1-0716-2140-0_9
