Biology Reference
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10. Sonicate the proteoliposome solution at room temperature for
18 s (10 % amplitude and 50 % duty cycle).
11. In order to remove unincorporated substrates, apply the pro-
teoliposome solution to a 5 cm bed-volume Dowex column.
Discard the fl ow-through.
12. Apply 300
l of equilibration solution to the 5 cm bed-volume
Dowex column and discard the fl ow-through.
13. Apply 500
μ
l of equilibration solution to the Dowex column
and collect the elution. This fraction contains the transport
assay proteoliposomes.
14. Divide the transport assay proteoliposome fraction into 100
μ
μ
l
of aliquots.
15. Store on ice until use (
see
Note 8
).
3.7 Transport
Assay of AtDTC
1. Pre-incubate 100
μ
l of transport assay proteoliposomes at
25 °C for 2 min.
2. Add 5
l of 2-oxoglutarate solution and mix gently.
3. Incubate at 25 °C for 0, 1, 5, 10, or 40 min.
4. Add 15
μ
l of stop solution and mix gently.
5. Apply the proteoliposome solution to a 4.2 cm bed-volume
Dowex column to eliminate any 2-oxoglutarate remaining
outside of the transport assay proteoliposomes. Discard the
fl ow-through.
6. Apply 700
μ
l of 200 mM sodium acetate to the column and
collect the elution. This fraction contains proteoliposomes.
7. The radioactivity associated with the eluted proteoliposomes is
measured with a liquid scintillation spectrometer.
8. The result of a typical AtDTC transport assay is shown in
Fig.
2b
.
μ
3.8 Preparation
of bR mRNA from
a PCR Reaction
In the following Subheadings
3.8
-
3.10
, a method for the synthesis
of integral MPs using WGCF system in the presence of mixed
detergents is described. Recently, we reported that a combination
of FC14 and CHAPS is effective for the WGCF synthesis and
purifi cation of detergent-solubilized bR in high yield [
3
].
Furthermore, bR synthesized and purifi ed from the mixed deter-
gent method was confi rmed to be functional by measuring the
decrease in fl uorescence intensity of acridine orange after reconsti-
tution into liposomes (data not shown).
1. Prepare a 50
μ
l of PCR reaction mixture as follows: 1
μ
l of tem-
plate plasmid (pEU3SH-bR [
3
], 1 ng/
μ
l), 1.5
μ
l of 10
μ
M pEU-
SPU primer (5
′
-ACATACGATTTAGGTGACACT-3
′
), 1.5
μ
l of
10
μ
M Anti3 primer (5
′
-GGAGAAAGGCGGACAGGTAT-3
′
),
4
μ
l of 2.5 mM dNTP mixture, 5
μ
l of 10×
Ex Taq
buffer, 0.25
μ
l
of
Ex Taq
polymerase, and 36.75
μ
l of Milli-Q water.
