Biology Reference
In-Depth Information
15.3.2
Step 2: purification of heterologously expressed wild-type
and azido-tagged rhodopsin
15.3.2.1
Materials
n
-Dodecyl-
b
-
D
-maltopyranoside (DM) (Affymetrix, Anatrace, D310, MW
¼
510.6 g/mol)
C9 peptide for specific elution (NH
2
-TETSQVAPA-COOH, synthesized by Bio
Basic)
1D4 Sepharose 2B resin (2 mg 1D4 monoclonal antibody/mL resin, prepared
by cyanogen bromide activation procedure) (
Oprian, Molday, Kaufman, &
Khorana
,
1987; Knepp, Grunbeck, Banerjee, Sakmar, & Huber, 2011
)
SOLUBILIZATION BUFFER: (
Sakmar, Franke, & Khorana, 1989
)
1% (w/v) DM
50 mM HEPES or Tris-HCl, pH 6.8
100 mM NaCl
1mMCaCl
2
With complete protease inhibitor cocktail (Roche)
REACTION AND WASH BUFFER:
0.1% (w/v) DM in Dulbecco's phosphate-buffered saline (DPBS), pH 7.2
ELUTION BUFFER:
0.33 mg/mL C9 peptide
0.1% (w/v) DM
2 mM phosphate buffer, pH 6.0
Centrifugation apparatus capable of centrifugation at 100,000
g
. We use a
Beckman Optima ultracentrifuge with a Beckman TLA100.3 rotor.
15 mL polypropylene Falcon tube for the solubilization process.
A deli-refrigerator installed in the dark room.
Lambda-800 spectrophotometer (PerkinElmer Life Sciences).
15.3.2.2
Regeneration of heterologously expressed opsin with
11-cis-retinal
Resuspend the harvested cells in DPBS (containing protease inhibitor) at a
density of 10
7
cells/mL in a 15 mL conical, polypropylene tube (Falcon).
Add 11-
cis
-retinal (1.4 mM ethanol solution) in the dark room into the cell
suspension to a final concentration of 5
M. Nutate the suspension at 4
C
m
overnight.
Remove the excess 11-
cis
-retinal by spinning down the cells and discard the
supernatant fraction. The regenerated cells can be immediately used or stored
at
20
C for several months.
15.3.2.3
Purification of rhodopsin
In order to prevent photobleaching of rhodopsin, always maintain the regenerated
cells and lysates in the dark.
