of the bleached area must be small enough so that the
unbleached protein is in excess, so that the contribution of
bleached protein in the exchange can be neglected. Alternatively, a whole-cell FRAP can be used to determine the rate of
new protein production [36].
5. While fixing the size of a bleach spot is important during each
series of experiments, due to the change in the diffusional
coefficient with bleach spot size and diameter [15, 23], a
comparison of the recovery when applying different bleach
spot sizes can be used to confirm and test the diffusional
component of the recovery. This is due to the half-time of the
recovery being affected by the spot size in the case of recovery
primarily through diffusion, but not when binding reactions
predominate over diffusion [23].
6. It is important to achieve a sufficient photobleaching depth
(the amount of signal lost relative to pre-bleach) for a meaningful recovery while avoiding overbleaching. If over 80% of
the initial signal is bleached, a Gaussian approximation of the
bleached region is no longer valid, meaning that most common
mathematical models cannot be applied [22, 28]. Additionally,
excessive bleaching might cause photodamage due to localized
heating [18, 37]. One approach which one can use to test for
any changes in protein dynamics due to photodamage is
sequential bleaching. Namely, to bleach an area, allow it to
recover, then bleach it again with the same parameters. In the
absence of photodamage, the recovery of the second bleach
event will have the same dynamics (number and half-times of
components) but will be close to complete (proportionally to
the extent of the first bleaching round) due to the immobile
fraction having already been bleached in the first round. This
control for photodamage has been applied by our group and
others [15, 38] and is an important consideration when starting a new FRAP experiment on a newly tagged protein or in a
new system. In our experience, the best means of producing
consistent results without photodamage is achieved when the
fluorescent protein is bleached to 20–40% of the initial fluorescent intensity. For E-cad-GFP, we use eight scans with 50–70%
laser power using a 488 nm wavelength laser.
7. The time to achieve sufficient bleaching must be minimized for
two reasons. First, one needs to consider that protein dynamics
do not cease while photobleaching is being performed, which
causes misinterpretation of the kinetics due to the corona effect
[39]. Longer bleaching times will lead to bigger bleached areas
due to the bleached protein moving out of the spot where laser
is applied. Secondly, localized heating mentioned above
increases with bleaching time, which could intensify potential
damage to cells and proteins [37]. Photobleaching times below
FRAP to Study the Dynamics of Proteins in vivo
155
unbleached protein is in excess, so that the contribution of
bleached protein in the exchange can be neglected. Alternatively, a whole-cell FRAP can be used to determine the rate of
new protein production [36].
5. While fixing the size of a bleach spot is important during each
series of experiments, due to the change in the diffusional
coefficient with bleach spot size and diameter [15, 23], a
comparison of the recovery when applying different bleach
spot sizes can be used to confirm and test the diffusional
component of the recovery. This is due to the half-time of the
recovery being affected by the spot size in the case of recovery
primarily through diffusion, but not when binding reactions
predominate over diffusion [23].
6. It is important to achieve a sufficient photobleaching depth
(the amount of signal lost relative to pre-bleach) for a meaningful recovery while avoiding overbleaching. If over 80% of
the initial signal is bleached, a Gaussian approximation of the
bleached region is no longer valid, meaning that most common
mathematical models cannot be applied [22, 28]. Additionally,
excessive bleaching might cause photodamage due to localized
heating [18, 37]. One approach which one can use to test for
any changes in protein dynamics due to photodamage is
sequential bleaching. Namely, to bleach an area, allow it to
recover, then bleach it again with the same parameters. In the
absence of photodamage, the recovery of the second bleach
event will have the same dynamics (number and half-times of
components) but will be close to complete (proportionally to
the extent of the first bleaching round) due to the immobile
fraction having already been bleached in the first round. This
control for photodamage has been applied by our group and
others [15, 38] and is an important consideration when starting a new FRAP experiment on a newly tagged protein or in a
new system. In our experience, the best means of producing
consistent results without photodamage is achieved when the
fluorescent protein is bleached to 20–40% of the initial fluorescent intensity. For E-cad-GFP, we use eight scans with 50–70%
laser power using a 488 nm wavelength laser.
7. The time to achieve sufficient bleaching must be minimized for
two reasons. First, one needs to consider that protein dynamics
do not cease while photobleaching is being performed, which
causes misinterpretation of the kinetics due to the corona effect
[39]. Longer bleaching times will lead to bigger bleached areas
due to the bleached protein moving out of the spot where laser
is applied. Secondly, localized heating mentioned above
increases with bleaching time, which could intensify potential
damage to cells and proteins [37]. Photobleaching times below
FRAP to Study the Dynamics of Proteins in vivo
155