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.
Keywords: CLSM; FRAP; Fluorescence recovery; Protein dynamics; SOX9; Transcription factor activity; mGFP.