Biology Reference
In-Depth Information
24.1.2
Methods
24.1.2.1
ROS isolation with sucrose gradient
The isolation of ROS from dark-adapted bovine retina essentially follows an estab-
lished sucrose density gradient centrifugation procedure (
Papermaster, 1982
) with
small modifications (
Salom, Li, Zhu, Sokal, & Palczewski, 2005
). Briefly, shake
the retinas for 1 min in 1 volume of 45% sucrose solution and centrifuge for
5 min at 3,300
g
. Filter the supernatant through a gauze-lined funnel, dilute it with
1 volume of Kuhn's buffer, and centrifuge for 10 min at 13,000
g
. Resuspend the
pellet from each tube in 1 mL of 1.10 g/mL sucrose plus 0.5 mL of Kuhn's buffer,
and load the suspension into tubes containing a three-step sucrose gradient (10, 16,
and 10 mL of densities 1.11, 1.13, and 1.15 g/mL, respectively). After centrifugation
for 20 min at 22,000
g
, collect the 1.11-1.13 g/mL interface, dilute it with 1 vol-
ume of Kuhn's buffer, and recover the ROS by centrifugation at 6,500
g
for 7 min.
80
C. Typical recovery is
Store the ROS pellet at
0.6 mg rhodopsin per retina.
24.1.2.2
Nonyl-glucoside/Zn(OAc)
2
extraction of rhodopsin
Rhodopsin can be selectively extracted from ROS membrane preparations by solu-
bilizing a ROS suspension with alkyl(thio)glucosides in the presence of 2B series
divalent cations, which eliminates opsin and other protein contaminants (
Okada,
Takeda, & Kouyama, 1998
).
Resuspend the ROS pellet in
1 volume of 50 mM MES, pH 6.35. Dissolve a
small aliquot in UV buffer and measure its absorbance spectrum from 250 to
700 nm to estimate the rhodopsin concentration (
e
498nm
ΒΌ
40,600 M
1
cm
1
;
Spalink, Reynolds, Rentzepis, Sperling, &Applebury, 1983
). Add the remaining sol-
ubilization components from their stock solutions to the ROS suspension to reach
final concentrations of 5-10 mg/mL of rhodopsin, 50 mM MES, pH 6.35,
100 mM Zn(OAc)
2
, and a NG/rhodopsin ratio of 2.2 (w/w). Mix briefly, incubate
overnight at 4
C, and remove precipitated proteins by centrifugation. The
A
280nm
/
A
500nm
ratio of the supernatant typically should be 1.8-2.0, indicating a rhodopsin
purity of 80-90%. Up to 50% of rhodopsin can be lost in this step.
24.1.2.3
Immunoaffinity purification of rhodopsin
Preparation of Sepharose-immobilized 1D4 monoclonal antibody is achieved by fol-
lowing the CNBr-activated Sepharose manufacturer's instructions. Antirhodopsin
1D4 antibody can be produced from hybridoma supernatant and purified with a
Diethylaminoethyl (DEAE)-cellulose or protein-A column. Alternatively, it can
be obtained from mouse ascites fluid and purified with T-Gel (Pierce). We typically
coupled the antibody at a ratio of 5 mg of 1D4 per mL of settled gel, resulting in a
yield of
0.5 mg purified rhodopsin per mL of gel.
This is a standard immunoaffinity chromatography step where solubilized ROS
(
0.6 mg rhodopsin per mL of settled gel) are slowly loaded onto a prepacked 1D4-
Sepharose column (10-20 min), the gel is washed with 5-10 column volumes of
washing buffer, and rhodopsin is recovered by addition of 0.5-1 mg/mL of
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