Biology Reference
In-Depth Information
interest; (3) and unlike copper-catalyzed azide-alkyne cycloaddition, it does not
involve any additional catalyst and thus is far less likely to damage the protein by
inducing undesirable radical reactions. So far, a series of cyclooctyne reagents have
been developed. We chose dibenzocyclooctyne (DIBO), which has been reported
for its superior bioorthogonality. In the first step, we tested this reaction for sites
in different regions of rhodopsin (Y102 in the second extracellular loop, M155 in
helix IV, and S144 in the second intracellular loop). We found that the resulting
stoichiometry is dependent on the site of labeling. We characterized the labeled
receptor in two ways: (1) UV-Vis spectroscopy to determine the stoichiometry
and (2) in-gel fluorescence to confirm the purity of the labeled product. We chose
Rho S144azF, a mutant that expresses at relatively high level (1
g/10
7
cells) and
m
reacts well with the cyclooctyne reagents (dye-to-protein ratio
1) to study the
kinetics of the reaction and examine the reactivity of DIBO reagents conjugated
to different Alexa fluorophores (Alexa 488, Alexa 555, Alexa 594, and Alexa
647). In-gel fluorescence was used to measure the degree of fluorescence labeling,
while silver staining was used to quantify the amount of protein.
15.4.1
Step 1: fluorescent labeling of azido-tagged rhodopsin
with SpAAC
15.4.1.1
Materials
Dibenzocyclooctyne reagents conjugated to Alexa fluorophores (Invitrogen,
dissolved in DMSO as 5 mM stock solution and stored at
20
C)
Thermomixer to control the temperature and shaking speed (Eppendorf)
15.4.1.2
Procedures
Immobilize the regenerated rhodopsin (expressed from a 30 mL
HEK293-F culture) to 100
L 1D4-mAb sepharose 2B as described in
m
Section 15.3.2.3
.
Transfer resin into a 1.5 mL Eppendorf tube and wash with the reaction buffer for
three times (30 min each time).
Mix the resin with 200
L slurry. Add
appropriate amount of labeling reagent into the slurry. Allow the reaction to
proceed at 25
C in the dark with agitation (shaking speed 900
L reaction buffer to give a 300
m
m
1000 rpm) for
desired reaction time.
Stop the reaction by centrifuging the resin and removing the supernatant fraction.
Wash the resin with the reaction/wash buffer to deplete the unreacted dyes.
Monitor the washing steps by recording the UV-Vis spectra of the wash buffer.
Based on our experience, five washes should be sufficient.
Elute the labeled receptor with the C9 peptide (100
m
L elution buffer
2).
Supplement the eluate with 150 mM NaCl.
