Biology Reference
In-Depth Information
Citric acid
Sodium hydroxide
30% ammonium hydroxide
38% formaldehyde (formalin)
Magnetic stirrer
A
B
ECL2
-
+
PNGaseF
Y102
TM4
75 kDa
M155
25 kDa
S144
C
D
M155-Alexa 555
Y102-Alexa 555
2.5
Dark
Light
Dark-light
Dark
Light
Dark-light
0.3
2.0
1.5
1.0
0.5
0.0
0.2
0.1
0.0
-
0.1
-
0.5
300
400
500
600
700
300
400
500
600
700
Wavelength (nm)
Wavelength (nm)
FIGURE 15.2
Fluorescent labeling of rhodopsin with Alexa 555-DIBO. (A) The tested sites are highlighted in
red in the crystal structure (PDB 1U19). (B) The corresponding in-gel fluorescence
images of the wild-type and azF-containing rhodopsin mutants labeled with Alexa 555-DIBO.
For all the three sites tested here, the initial concentration of Alexa 555-DIBO was 50
m
M.
Following SDS-PAGE electrophoresis, the image of the gel was acquired with a confocal
fluorescence scanner using 532 nm laser. The absence of fluorescent band for wt rhodopsin
treated under the same condition indicated that this reaction is specific for azF. PNGaseF
treatment of Rho S144-Alexa 555 resolved the monomer, dimer, and higher oligomer bands.
(C) The dark-state, light-state (after photobleaching), and difference (dark-light) spectra of
Rho M155-Alexa 555. The label-to-protein ratio was calculated from the 555 nm absorbance
in the green spectrum and the 500 nm absorbance in the difference spectrum (blue). The
extinction coefficients used for Alexa 555 and rhodopsin is 155,000 M
1
cm
1
and
40,500 M
1
cm
1
, respectively. The dye-to-protein ratio for Rho M155-Alexa 555 was
calculated to be 1.00. (D) The dark-state, light-state, and difference spectra of Rho
Y102-Alexa 555. The dye-to-protein ratio was calculated to be 0.44.
