Biology Reference
In-Depth Information
The regenerated cells were lysed with the solubilization buffer for at least 1 h at 4
C.
Centrifuge the cell lysates at 100,000
g
for 30 min at 4
C to clear the cell
lysate.
Mix the supernatant fraction with 50 or 100
L 1D4-mAb sepharose 2B and
m
incubated overnight at 4
C.
Transfer the resin into a Millipore Ultrafree-MC centrifugal filtering unit (pore
size 0.45
m), which allows for easy removal of wash buffer.
Wash the resin with the wash buffer for three times (30 min each time) and then
once with the elution buffer depleted of the C9 peptide to remove the salt.
Elute the receptor with 100
m
L (no less than the volume of the packed beads)
m
elution buffer.
Incubate the resin with the elution buffer at 4
C for at least 1 h.
Collect the purified receptor in a clean 1.5 mL Eppendorf tube by centrifugation.
Two elutions should recover 70-80% of the receptor.
Add 150 mM NaCl to the sample to give appropriate ionic strength. If no further
experiment follows immediately, keep the eluates at
80
C and thaw on ice
before use.
15.3.2.4
Characterization of purified rhodopsin with UV-Vis spectroscopy
Record the absorption spectrum of rhodopsin on a Lambda-800 spectrophotometer
(PerkinElmer Life Sciences) in a cuvette with a 10 mm path length. We
recommend using a cuvette suitable for samples of a small volume.
First obtain the dark spectrum of rhodopsin. Make sure the sample has
not been exposed to light source that causes the photoisomerization of
11-
cis
-retinal.
To acquire the light spectrum, photobleach the receptor by irradiating the sample
with a 505 nm LED light source (Thorlabs) and adding 25 mM NH
2
OH to cleave
the Schiff-base bond between opsin and retinal. Record the spectrum.
Calculate the difference spectrum (dark spectrum-light spectrum).
Determine the concentration of rhodopsin based on the intensity of the 500 nm
peak in the difference spectrum (assuming an extinction coefficient of
40,600 M
1
cm
1
at A
500
). The yield for heterologously expressed wt rhodopsin
normally ranges from 5 to 8 g per 10
7
cells. The yield for amber codon
suppression is
g per 10
7
cells, but it is dependent on the site of mutation.
0.5-1
m
15.4
PROTOCOL 2: LABELING WITH FLUORESCENT PROBES
USING CYCLOOCTYNES
Strain-promoted azide-alkyne cycloaddition (SpAAC) is chosen to label the azido
group incorporated into rhodopsin. This chemistry has the following advantages that
make it suitable for our specific purpose: (1) it exhibits good bioorthogonality, that
is, reacts specifically to azido group rather than to other reactive groups occurring in
the natural amino acids; (2) its fast kinetics enables robust labeling of the protein of
