Using FRAP to quantify changes in transcription factor dynamics after cell stimulation: Cell culture, FRAP, data analysis, and visualization

Kannan Govindaraj, Janine N. Post*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

Abstract

Here we show how to measure the mobility of transcription factors using fluorescence recovery after photobleaching (FRAP). Transcription factors are DNA-binding proteins that, upon binding to specific DNA motifs, regulate transcription of their target genes. FRAP is a simple, fast, and cost-effective method, and is a widely used quantitative method to measure the dynamics of fluorescently labeled molecules in solution, membranes, and inside living cells. Dynamics, specified by the immobile fraction, recovery half-time, diffusion constant, and ratio of molecules contributing to different phases of FRAP recovery, can be quantified by FRAP. This can be useful to understand their function in gene regulation. This tutorial is intended to familiarize the reader with the FRAP procedure to quantify transcription factor dynamics using a standard confocal microscope and analysis using MATLAB (MathWorks®). This article will guide the reader through the preconditions of FRAP, and a detailed and step-by-step procedure of preparing cells, bleaching protocol, data analysis in MATLAB, and visualization of the FRAP data.

Original languageEnglish
Title of host publicationOsteoporosis and Osteoarthritis
PublisherHumana Press Inc.
Pages109-139
Number of pages31
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2221
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • CLSM
  • Fluorescence recovery
  • FRAP
  • mGFP
  • Protein dynamics
  • SOX9
  • Transcription factor activity

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