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Analysis of Protein Kinetics Using Fluorescence Recovery After Photobleaching (FRAP)

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1563))

Abstract

Fluorescence recovery after photobleaching (FRAP) is a cutting-edge live-cell functional imaging technique that enables the exploration of protein dynamics in individual cells and thus permits the elucidation of protein mobility, function, and interactions at a single-cell level. During a typical FRAP experiment, fluorescent molecules in a defined region of interest within the cell are bleached by a short and powerful laser pulse, while the recovery of the fluorescence in the region is monitored over time by time-lapse microscopy. FRAP experimental setup and image acquisition involve a number of steps that need to be carefully executed to avoid technical artifacts. Equally important is the subsequent computational analysis of FRAP raw data, to derive quantitative information on protein diffusion and binding parameters. Here we present an integrated in vivo and in silico protocol for the analysis of protein kinetics using FRAP. We focus on the most commonly encountered challenges and technical or computational pitfalls and their troubleshooting so that valid and robust insight into protein dynamics within living cells is gained.

*These authors contributed equally to this work.

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Acknowledgments

We thank the Advanced Light Microscopy Facility of the University of Patras for assistance with live-cell imaging experiments and all members of the Cell Cycle and Stem Cell labs, Medical School, University of Patras for helpful discussions.

Work in our lab is supported by a grant from the European Research Council (DYNACOM, 281851).

Appendix

Let y(t)ROI1, y(t)ROI2, and y(t)ROI3 represent the fluorescence intensity in the bleaching region, the total area of fluorescence, and a random background area. Background correction is performed by simply subtracting the background measurements from the rest:

$$ \begin{array}{c} y{(t)}_{ROI1*}= y(t){}_{ROI1}- y(t){}_{ROI3}\\ {} y{(t)}_{ROI2*}= y(t){}_{ROI2}- y(t){}_{ROI3}\end{array} $$

Let \( {y}_{ROI1}^{pre} \) denote the average intensity in the bleaching region during the pre-bleach interval and t bleach + 1 denote the first time point after the bleach. Bleaching depth (bd) is computed as follows:

\( bd=\frac{y_{ROI1}^{pre}- y{\left({t}_{\mathrm{bleach}+1}\right)}_{ROI1*}}{y_{ROI1}^{pre}} \) (1)

Similarly, let \( {y}_{ROI2}^{pre},{y}_{ROI2}^{\mathrm{post}} \) denote the average intensities in the total area of fluorescence during the pre- and post-bleach interval respectively. Gap ratio (gr) is computed as follows:

\( gr=\frac{y_{ROI2}^{\mathrm{post}}}{y_{ROI2}^{pre}} \) (2)

Double normalization is computed as follows:

\( y{(t)}_{\mathrm{double}}=\frac{y{(t)}_{ROI1*}}{y_{ROI1}^{pre}}\times \frac{y_{ROI2}^{pre}}{\mathrm{y}{(t)}_{ROI2*}} \) (3)

Similarly, full-scale normalization is computed as follows:

\( y{(t)}_{\mathrm{fullscale}}=\frac{y{(t)}_{\mathrm{double}}- y{\left({t}_{\mathrm{bleach}+1}\right)}_{\mathrm{double}}}{1- y{\left({t}_{\mathrm{bleach}+1}\right)}_{\mathrm{double}}} \) (4)

To compute quantitative parameters such as t-half (t 1/2) and mobile fraction (F mob ), only the post-bleach part of the curve is necessary. Dropping normalization index for simplicity, let y(t end) denote the normalized intensity (double or full-scale) when the curve has reached its plateau and y(t bleach + 1) denote the first post-bleach measurement. To remove the pre-bleach part of the curve from the measurements, we simply subtract t bleach + 1 from the rest of the time points, leading to y(t bleach + 1) = y(t = 0). It is:

\( {F}_{mob}=\frac{y\left({t}_{end}\right)- y\left( t=0\right)}{1- y\left( t=0\right)} \) (5)

Immobile fraction (F imm) is defined as the fraction of bleached molecules that were bound and do not diffuse away from the bleaching area by the end of the experiment. It is:

\( {F}_{imm}=\frac{1- y\left({t}_{end}\right)}{1- y\left( t=0\right)} \) (6)

Naturally, F mob + F imm = 1. It is clear that for curves that exhibit full recovery, y(t end) = 1, leading to F imm = 0 and F imm = 1.

The value of t 1/2 is computed as follows:

\( y\left({t}_{1/2}\right)=\frac{y\left({t}_{end}\right)+ y\left( t=0\right)}{2} \) (7)

To estimate the values of these parameters, the experimental data are fitted to one of the following exponential equations:

y(t)single = y 0 − αe −βt(8)

y(t)double = y 0 − αe −βt − γe −δt(9)

If full-scale normalization was used, then it is: y(t = 0) = 0 and from Eq. (5) it is F mob = y(t end) = y 0, since for both Eqs. (8) and (9) as t → ∞,  y(t end) = y 0. For double normalization, we have:

  1. 1.

    Using single exponential fitting (Eq. (8)) it is y(t = 0)single = y 0 − a,and from Eq. (5) it is:

$$ {F}_{mob}=\frac{y_0-{y}_0+ a}{1-{y}_0+ a}=\frac{a}{1-{y}_0+ a} $$
  1. 2.

    Using double exponential fitting (Eq. (9)) it is y(t = 0)double = y 0 − a − γ, and again from Eq. (5) it is:

$$ {F}_{mob}=\frac{y_0-{y}_0+ a+\gamma}{1-{y}_0+ a+\gamma}=\frac{a+\gamma}{1-{y}_0+ a+\gamma} $$

The value of t 1/2 is estimated from Eq. (7) as follows:

  1. 1.

    Using single exponential fitting and since similarly as above y(t = 0)single = y 0 − a

$$ \begin{array}{c}\frac{y_0+{y}_0- a}{2}={y}_0-\alpha {e}^{-\beta {t}_{1/2}}\\ {}{e}^{-\beta {t}_{1/2}}=\frac{1}{2}\\ {}{t}_{1/2}=\frac{ \ln 2}{\beta}\end{array} $$
  1. 2.

    Using a double exponential fitting equation, the value of t 1/2 is estimated numerically, since there is no closed form solution.

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    Authors and Affiliations

    1. Laboratory of Biology, School of Medicine, University of Patras, GR26500 Rio, Patras, Greece

      Nickolaos Nikiforos Giakoumakis, Maria Anna Rapsomaniki & Zoi Lygerou

    2. IBM Research Zurich, Säumerstrasse 4, CH-8803, Rüschlikon, Switzerland

      Maria Anna Rapsomaniki

    Authors
    1. Nickolaos Nikiforos Giakoumakis
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    2. Maria Anna Rapsomaniki
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    3. Zoi Lygerou
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    Correspondence to Zoi Lygerou .

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    Editors and Affiliations

    1. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California, USA

      Yolanda Markaki

    2. Biozentrum der LMU Munchen, Planegg-Martinsried, Germany

      Hartmann Harz

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    Giakoumakis, N.N., Rapsomaniki, M.A., Lygerou, Z. (2017). Analysis of Protein Kinetics Using Fluorescence Recovery After Photobleaching (FRAP). In: Markaki, Y., Harz, H. (eds) Light Microscopy. Methods in Molecular Biology, vol 1563. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6810-7_16

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    • DOI: https://doi.org/10.1007/978-1-4939-6810-7_16

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6808-4

    • Online ISBN: 978-1-4939-6810-7

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