FLUORESCENT RECOVERY AFTER
PHOTOBLEACHING
Analysis and Applications
Zareen Butt
Department of Chemistry and Biochemistry
University of Windsor
OVERVIEW
 Phenomenon of Photobleaching
 Fluorescent Recovery After Photobleaching
 Measure the mobility of nuclear
proteins, macromolecular diffusion within cell
membranes, the cytoplasm, nucleoplasm
HOW FRAP WORKS
 Molecules covalently bound to a fluorophore
 Inhomogenous fluorescent population
 Spatial separation between fluorescent moleclues
and photobleached molecules at time 0
Photobleached
Population
Example of photobleaching in an indian muntjac
fibroblast nucleus expressing ASF/SF2 GFP
DATA COLLECTION
0s
20s
30s
10s
90s
FLUORESCENT RECOVERY CURVE
Intensity
Intensity
Time (seconds)
Nucleoplasmic
Chromatin
associated
Transcription
Factor
Topoisomerase I &
Topoisomerase IIα
&ß
Nucleosomal
Histones
Estrogen Receptor
Glucocorticoid
Receptor
Nuclear
Emerin
lamina/memebrane HA-95
List of some nuclear proteins investigated by FRAP
PREFRAP ANALYSIS
 Steady-State distribution in living cells
Artifactual Distributions
 Diffused distribution
 Formation of large spherical aggregates
 Examples of these distributions illustrated for
histone deactylase-GFP fusion proteins
Abnormal distribution of GFP-tagged histone
deacetylases in mouse 10t1/2 cells transfected with
HDAC4-GFP (left) and HDAC3-GFP (right).
DATA NORMALIZATION
 The raw data must be normalized in order
to compensate for:
1) the background signal in the image
2) the loss of total cellular
fluorescence due to photobleaching
a subregion of the cell
3) any loss of fluorescence that
occurs during the course of collection
of recovery time series
DATA ANALYSIS
 Diffusion coefficient (measures the rate of
movement and represents the mean squared
displacement of proteins over time)

Effective diffusion coefficient (does not take into
consideration any interaction the proteins might
undergo in the process of diffusion)
RECENT ENZYMATIC STUDIES USING
FRAP ANALYSIS
 gp130/Jak 1 interaction
Kinetics of association and the state of activation of
GTPases in phagosomes
Mobility of Glucocorticoid Receptor in the nucleus
Phospholipase C-β2 activity and mode of
memebrane interactions in living cells
FRAP analysis of gp130-YFP at the plasma
membrane
FRAP ANALYSIS OF gp-130YFP AT
THE PLASMA MEMBRANE
 Cos-7
cells transfected with a gp130-YFP
containing expression vector
 Region of interest with a diameter of 1.3µm is
photobleached
 As a result of double bleaching, the mobile and
immobile fractions remains constant
The fraction of mobile and immobile fractions
remains constant after double bleaching
FRAP recovery curves demonstarting that Rac
2(12V) reduces fluorescent recovery rate of GFPPLCß2
Rab-GFP FRAP.
RECENT ENZYMATIC STUDIES USING
FRAP ANALYSIS
 gp130/Jak 1 interaction
 Kinetics of association and the state of activation of
GTPases in phagosomes
 Mobility of Glucocorticoid Receptor in the nucleus
 Phospholipase C-β2 activity and mode of
memebrane interactions in living cells
CONCLUSION
In the future, FRAP combined with useful
mathematical analysis, and use of engineered
proteins will serve as an important tool to study the
mobility of molecules in living cells
REFERENCES
 Carrero, G., Macdonald, D., Crawford, E., Vries
de., and Hendzel, M. (2003) Methods. 29, 14-28
 Giese, B., Au-Yeung, C., Herrmann, A.,
Diefenbach, S., Haan, C., Kuster,A., Wortmann S.,
Roderburg, C., Heinrich P., Behrmann, I., and
Muller-Newen, G. (2003) The journal of
biochemistry. 278, 39205-39213
 Illenberger, C., Walliser, C., Strobel, J., Gutman,
O., Niv, H., Gaidzik, V., Kloog Y., Gierschik, P., and
Henis, Y. (2003) The journal of biochemistry. 278,
8645-8652
 Schaaf, M., and Cidlowski, J. (2003) Molecular and
Cellular Biology. 23, 1922-1934
 Vieira, O., Bucci, C., Harrison, R., Trimble, W.,
Lanzetti, L., Greunberg J., Schreiber, A., Stahl, P.,
and Grinstein, S. (2003) Molecular and Cellular
Biology. 23, 2501-2514