Fluorescence Recovery After Photobleaching (FRAP) with a Focus on F‐actin

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

In this unit on fluorescence recovery after photobleaching (FRAP), an imaging approach to study protein?protein interactions in situ is described. The protocols presented use confocal microscopy to examine the mobility of a fluorescent protein by measuring the recovery of the protein in a bleached area. The data gained in FRAP studies is qualitative and yields insight into relative binding affinity, binding characteristics, and the effect of treatments or mutations on protein mobility. Curr. Protoc. Neurosci. 61:2.17.1?2.17.12. © 2012 by John Wiley & Sons, Inc.

Keywords: FRAP; actin; protein mobility; GFP

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Introduction
Basic Protocol 1: FRAP Imaging of Fluorescently Tagged Actin in Cells
Basic Protocol 2: Analysis of the FRAP Time‐Lapse Image Series
Basic Protocol 3: FRAP of Immobilized Actin
Basic Protocol 4: Determination of Diffusion‐Coupled or Diffusion‐Uncoupled Recovery
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: FRAP Imaging of Fluorescently Tagged Actin in Cells

Materials

Cells of choice (cell lines, primary cell cultures, etc.)
Medium of choice
Fluorescently tagged actin or protein of interest
Culture external base (CEB; see recipe )

Glass‐bottom dishes, 35 mm
Microscope eguipped with epifluorescence capability
Confocal laser‐scanning microscope equipped with photobleaching protocols

Basic Protocol 2: Analysis of the FRAP Time‐Lapse Image Series

Materials

Time‐lapse image series from a photobleaching experiment ( protocol 1 )
Image analysis software (typically part of the confocal microscope software)
Data analysis software (e.g., Microsoft Office Excel or Origin 6.1)

Basic Protocol 3: FRAP of Immobilized Actin

Materials

Cells expressing tagged actin, cultured on 35‐mm glass‐bottom dish ( protocol 1 )
Culture external base (CEB; see recipe )
Actin‐immobilizing agent (cytochalasin D or jasplakinolide)

Microscope equipped with epifluorescence capability
Confocal laser‐scanning microscope equipped with photobleaching protocols

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Figures

Figure 2.17.1 Schematic illustrating FRAP. A cell expressing fluorescently tagged protein undergoes high‐power laser photobleaching in a defined region of interest (ROI) in the cell. Recovery from the laser bleach is monitored over time by low‐power laser imaging. Due to imaging there is a reduction in whole‐cell fluorescence concurrent with a recovery of fluorescence in the bleached ROI.

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Figure 2.17.2 Fluorescence recovery curve. The data have been normalized for background reduction in whole‐cell fluorescence and normalized to prebleach fluorescence intensity [ F (−1)]. Immobile and mobile fractions are indicated. F (−1) = prebleach background‐normalized fluorescence in the ROI at t = −1. F (∞) = final recovered fluorescence value in the ROI that is a constant value or plateau (recovery is considered complete at F (∞)). F (0) = fluorescence value at time zero after the bleach. t ₀ = the first time point after the bleach. t _1/2 = half‐time of fluorescence recovery, i.e., the time required to reach 50% of final recovered fluorescence.

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Figure 2.17.3 FRAP of protein after immobilization of actin. The fluorescence recovery of an actin‐interacting protein in untreated control (black line), after 1 µM jasplakinolide treatment (JS, dark gray line), and after 10 µg/ml cytochalasin D treatment (CD, light gray line) is shown. Reprinted with permission from Lin and Redmond; © 2008 National Academy of Sciences, U.S.A

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Literature Cited

Literature Cited
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