Biology Reference
In-Depth Information
ending with the 15 % sucrose solution. The separation between
each sucrose step should be easily visible to the naked eye due
to the refringence difference. Cell lysate containing the dodeca-
hedron (cf:
step 5
of Subheading
3.1
) can then be loaded onto
the top of the gradient.
3. Insert both the sample and the balance tubes in previously
refrigerated SW41buckets, then equilibrate them accurately
with HBS-G buffer. These buckets are then placed in the SW41
rotor together with empty buckets at the remaining positions.
Centrifugation is then run for 18 h at 4 °C at 280,000 ×
g
.
4. At the end of the run, the ultraclear tube is recovered from the
bucket. Fractionation is then done from top to bottom by tak-
ing 850
L samples from the top of the upper solution until
the bottom is reached (around 14 fractions as shown in Fig.
2
)
(
see
Note 5
).
5. Dodecahedron-containing fractions (usually 11-15) are then
pooled together and dialyzed against HBS buffer.
μ
Fig. 2
Dodecahedron purifi cation.
Upper panel
: SDS-PAGE analysis of a portion of
each fraction, from top to bottom gradient, after fractionation (
Bs
Base,
Fb
Fiber)
Dd is found in bottom fraction.
Lower panels
: Quality control by electron micros-
copy negative staining of Pt-Dd (
Left
) and Bs-Dd (
right
)
