Biology Reference
In-Depth Information
4. Overlay 650
μ
l of 35 % Accudenz solution, 650
μ
l of 30 %
l of ultracentrifugation solution.
5. Centrifuge the density gradient tube for 1 h at 206,000 ×
g
and
4 °C.
6. Fractionate the density gradient by carefully removing 200
Accudenz solution, and 100
μ
l of
aliquots from the top to the bottom of the tube (
see
Note 10
).
7. To determine which fractions contain protein, subject fractions
to SDS-PAGE and detect the [
14
C]-labeled AtCBL1 with imag-
ing analyzer as described above (Subheading
3.12
) (Fig.
4b
).
μ
4
Notes
1. All solutions are prepared using Milli-Q water prepared by
Autopure WT101UV (Yamato Scientifi c Co., Ltd., Tokyo,
Japan) with BioPak fi lter (Nihon Millipore K.K., Tokyo,
Japan), in order to eliminate deoxyribonucleases, RNases, and
pyrogens.
2. The white pellet is magnesium pyrophosphate, a by-product of
mRNA synthesis.
3. We freshly prepare liposomes for each experiment and store
them on ice until use.
4. The Dowex resin can be stored at 4 °C until use.
5. Micro spatulas are useful for plugging the end of Pasture
pipettes with absorbent cotton.
6. The synthesized MPs co-sediment with liposomes by
centrifugation.
7. These proteoliposomes can be stored at −80 °C for at least
1 week.
8. We use these proteoliposomes for transport assay within 1 h.
9. Before use, wash the inside of the dialysis cups and receptacle
tubes with Milli-Q water. Ensure that dialysis cup does not
leak. Receptacle tubes can be autoclaved and reused.
10. After ultracentrifugation, liposomes are found at the interface
between the 30 % Accudenz and ultracentrifugation solution
layers.
References
1. Nozawa A, Nanamiya H, Miyata T et al (2007)
A cell-free translation and proteoliposome
reconstitution system for functional analysis of
plant solute transporters. Plant Cell Physiol
48:1815-1820
2. Nozawa A, Fujimoto R, Matsuoka H et al
(2011) Cell-free synthesis, reconstitution, and
characterization of a mitochondrial dicarbo-
xylate-tricarboxylate carrier of Plasmodium fal-
ciparum. Biochem Biophys Res Commun
414:612-617
3. Genji T, Nozawa A, Tozawa Y (2010) Effi cient
production and purifi cation of functional
bacteriorhodopsin with a wheat-germ cell-free
