Biology Reference
In-Depth Information
fluorescence spots,
10 nm), and (iii) FRET
(3-10 nm). Multicolor methods have the unique advantage to allow simultaneous
observation of compositional and conformational dynamics of macromolecular
complexes. Automated SMD-TIRF microscopy facilitates systematic studies of
biochemical parameters, such as concentration, pH, ionic strength, and osmotic
pressure (and molecular crowding). For example, two concentrations have to be
systematically varied in the study of three-component (ternary) complexes that
are characteristic for the allosteric GPCR signalosomes (
Huber & Sakmar, 2011
).
(ii) dual-color colocalization (
>
15.3
PROTOCOL 1: GENETICALLY ENCODED AZIDO GROUPS
FOR BIOORTHOGONAL CONJUGATION
We have established a robust transient transfection protocol for amber codon sup-
pression in HEK293-T adherent cells. However, the adherent cells are cumbersome
for scaling up. We have adapted our transient transfection protocol for HEK293-F
suspension cells, which greatly reduced our time invested on protein expression.
We have incorporated an unnatural amino acid
p
-azido-
L
-phenylalanine (azF) into
rhodopsin and shown that both wild-type and mutant rhodopsin could be regenerated
and purified from suspension cells. UV-Vis spectroscopy can characterize the puri-
fied rhodopsin. The expression level of UAA-containing rhodopsin in the suspension
cells is comparable to that of the adherent cells.
15.3.1
Step 1: transfection of the HEK293-F suspension cells
to express azido-group tagged opsin
15.3.1.1
Materials
FreeStyle
™
293-F Cells (Invitrogen, R790-07)
Gibco
®
FreeStyle
™
293 Expression Medium (Invitrogen, 12338018)
FreeStyle
™
MAX Reagent (Invitrogen, 16447-100)
OptiPRO
™
SFM (Invitrogen, 12309-019)
p
-azido-
L
-phenylalanine (Chem-Impex International, 06162, MW
¼
206.1 g/mol)
125-mL polycarbonate, disposable, sterile Erlenmeyer flasks
Millipore Steriflip-GP filter unit, 0.22
m (Millipore, SCGP00525)
Sterile Eppendorf tube and conical, polycarbonate centrifuge tube (15 and 50 mL)
11-
cis
-retinal (1.4 mM stock solution dissolved in ethanol)
5% CO
2
cell culture incubator (Forma Scientific)
Platform shaker for growing suspension cell culture (New Brunswick Scientific,
Innova 2000)
Room temperature tabletop centrifuge
A dark room for handling light-sensitive materials
m
PLASMIDS FOR TRANSFECTION:
1.
pSVB.Yam carrying the gene encoding an amber suppressor tRNA
2.
pcDNA.RS carrying the tRNA synthetase for azF
