Biology Reference
In-Depth Information
FIGURE 24.1
Immunoaffinity purification of bovine rhodopsin from ROS solubilized in NG. (A) Purified
rhodopsin was eluted from a 1.5 cm
30 cm column containing 46 mL of 1D4-Sepharose.
(B) Absorption spectrum of purified rhodopsin (an aliquot was diluted in UV buffer).
(C) Coomassie-stained SDS-PAGE gel of purified rhodopsin. MW markers (SeeBlue ®
Plus2, Invitrogen) are shown in the right lane.
competing peptide. The first fractions with rhodopsin start eluting at
0.85 column
volumes with the peak fraction at
1.0 column volume ( Fig. 24.1 A). Elution can be
achieved in 1-2 h, but to maximize the peak concentration (up to 6 mg/mL), we typ-
ically carried out the elution with 1.5 column volumes of elution buffer for 4 h in a
20-50 cm long column ( Salom, Le Trong, et al., 2006 ). Rhodopsin's purity, assessed
by the A 280nm / A 500nm ratio and electrophoresis, is
99% ( Fig. 24.1 B and C).
Aliquots of fractions are mixed with UV buffer to measure their rhodopsin absor-
bance, and those fractions with the highest absorbance are pooled to achieve a rho-
dopsin concentration of 1-2 mg/mL. Up to 90% of rhodopsin loaded onto the column
can be recovered for the next step.
>
24.1.2.4 (NH 4 ) 2 SO 4 -induced phase separation
Addition of (NH 4 ) 2 SO 4 at saturating concentrations to NG solutions induces a phase
separation with a detergent-rich top phase. When rhodopsin purified in NG is treated
with (NH 4 ) 2 SO 4 , the bottom aqueous phase appears completely colorless and rho-
dopsin can be effectively concentrated up to 25-fold in the top phase ( Fig. 24.2 A).
Search WWH ::




Custom Search