Biology Reference
In-Depth Information
15.4.2
Step 2: characterization of labeled receptor using UV-Vis
spectroscopy and in-gel fluorescence
15.4.2.1
Material
Lambda-800 spectrophotometer (PerkinElmer Life Sciences)
Apparatus for SDS-PAGE electrophoresis
Confocal Typhoon 9400 fluorescence scanner (GE Healthcare, Life Sciences)
15.4.2.2
UV-Vis spectroscopy
The acquisition of the dark, light (photobleached), and difference spectra for
labeled rhodopsin is performed as described for unlabeled samples in Section
15.3.2.4. Results for samples labeled at different positions and with different fluor-
ophores are shown in
Figs. 15.2 and 15.3
. Determine the labeling stoichiometry (dye-
to-protein ratio) from the maximal absorption peak of Alexa Fluors (
A
fluor
) in the
light spectrum and from the rhodopsin absorption at 500 nm (
A
Rho, 500
) in the differ-
ence spectrum:
A
flour
=e
flour
A
Rho
;
500
=e
Rho
;
500
The extinction coefficients used for the calculation are listed in the succeeding table:
c
fluor
c
Rho
¼
Labeling stoichiometry
¼
Alexa
488-DIBO
Alexa
555-DIBO
Alexa
594-DIBO
Alexa
647-DIBO
Chromophore
Rho
1
1
e/M
cm
40,600
71,000
155,000
92,000
239,000
15.4.2.3
In-gel fluorescence
Load 50-100 ng of the labeled rhodopsin under reducing conditions to
SDS-PAGE gel. There is no boiling treatment of the sample prior to loading so as
to reduce protein aggregation.
Wash the gels briefly in ultrapure water.
Visualize the gel on a confocal Typhoon 9400 fluorescence scanner. For Alexa
488, 488 nm laser is used as the excitation light and the emission light is filtered
with a 520
10-nm band-pass. For Alexa 555, 532 nm laser and 580
15 nm
band-pass are used.
15.4.3
Step 3: kinetic study of the SpAAC
15.4.3.1
Materials for silver staining:
Acetic acid
Methanol
Ultrapure water (Milli Q Synthesis)
Silver nitrate (powder)
