Biology Reference
In-Depth Information
S144azF+Alexa 488
S144azF+Alexa 555
S144azF+Alexa 594
S144azF+Alexa 647
Rho
Rho
Rho
Rho
S144azF Rho
S144azF Rho
S144azF Rho
S144azF Rho
0.8
0.8
1.2
0.6
0.6
0.6
0.8
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
0.0
0.0
0.8
Dark
Light
Dark-light
Dark
Light
Dark-light
Dark
Light
Dark-light
Dark
Light
Dark-light
0.8
0.6
0.4
0.2
0.0
-0.2
1.6
0.8
0.6
1.2
0.6
0.4
0.8
0.4
0.2
0.4
0.2
0.0
0.0
0.0
300
400
500
600
300
400
500
600
700
300
400
500
600
700
300
400
500
600
700
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
FIGURE 15.3
Fluorescent labeling of azF-containing rhodopsin at site S144 with Alexa Fluor-DIBO. Upper panel: the normalized spectra for Rho S144-Alexa
Fluor and Rho wt treated under the same conditions. Lower panel: the dark-state, light-state, and difference UV-visible spectra rhodopsin
labeled at S144. The initial concentration of Alexa 488-DIBO is 5
M, for Alexa 555-DIBO 50
M, for Alexa 594-DIBO 5
M, and for Alexa
m
m
m
647-DIBO 20
M. The extinction coefficients for Alexa 488-DIBO, Alexa 555-DIBO, Alexa 594-DIBO, and Alexa 647-DIBO are assumed to be
71,000 M
1
cm
1
, 155,000 M
1
cm
1
, 92,000 M
1
cm
1
, and 239,000 M
1
cm
1
. The apparent dye-to-protein ratios for Rho S144-Alexa
488, Rho S144-Alexa 555, Rho S144-Alexa 594, Rho S144-Alexa 647 are 1.13, 1.11, 1.20, and 1.03, respectively.
m
