Biology Reference
In-Depth Information
Fig. 2
Synthesis and characterization of AtDTC. (
a
) Detection of synthesized
AtDTC proteins by CBB staining. The proteins were synthesized by liposome-
supplemented WGCF system. After protein synthesis, the reaction mixture was
centrifuged at 20,000 ×
g
for 20 min at 4 °C. The total (T), supernatant (S), and
pellet (P) fractions were subjected to SDS-PAGE and analyzed by CBB staining.
(
b
) Time course of the incorporation of external substrate [
14
C]2-oxoglutarate into
proteoliposomes reconstituted with AtDTC. AtDTC was synthesized by liposome-
supplemented WGCF system. The liposomes and synthesized protein complexes
were sedimented by centrifugation, and reconstituted into proteoliposomes
preloaded with 30 mM of 2-oxoglutarate. Intake of [
14
C]2-oxoglutarate was
measured (reproduced from ref.
2
)
electrophoresis (SDS-PAGE) and Coomassie brilliant blue
(CBB) staining (Fig.
2a
).
6. Mix 150
μ
l of the sonicated resuspended pellet with 150
μ
l of
substrate-preloaded liposomes.
7. Sonicate the mixed liposomes at room temperature for 18 s
(10 % amplitude and 50 % duty cycle). This is the proteolipo-
some solution.
8. Freeze the proteoliposome solution with liquid nitrogen
(
see
Note 7
).
9. Thaw the proteoliposome solution at room temperature.
