Biology Reference
In-Depth Information
adding a tube (MWCO 6-8 kDa) with 1 mg/mL BSA together
with the tube-containing labeled dodecahedron. The free dye
molecules escaping from the Dd tube will rapidly be recap-
tured and fi xed by BSA hence maintaining a low concentration
of dye in the buffer and then displacing the equilibrium
between inside and outside the tube. This produces a dye-
labeled BSA stock.
4. Be gentle when recovering Dd from cell. Centrifuge at low
speed (600 ×
g
) to avoid cell lysis that would result in protein
release in the supernatant. Only 2-3 mL can be loaded onto
the gradient avoiding the use of supernatant as a sample source.
5. Aliquots of 15
L of each fraction can be taken to check the
presence of the protein of interest (penton base and fi ber) by
standard SDS-PAGE procedure and Coomassie staining (
see
Fig.
2
).
6. The Ad3 penton base monomer weighs 60,000 Da, which is
100× more than the average dye weight (600 Da). The fi nal
concentration of the dye is ten times less than the dodecahe-
dron one, meaning ten dyes per monomer. Twenty-fi ve out of
the 544 amino acids of the Ad3 penton base are lysines with
free amines in the lateral chain. The maximum expected yield
is thus (10/25 = 40 %) of labeled lysines per monomer. So
fi nally, the dodecahedron can carry a theoretical maximum of
600 dyes per particle (10 × 5 × 12), giving a good compromise
between brightness and non-neutralizing activity of the dye.
7. “Tip” to limit toxicity: Observation can be done using medium
with or without serum, PBS is not recommended. When pos-
sible, use the highest possible wavelength (i.e., infrared), which
results in lower phototoxicity. NB: No toxicity has been
observed for Dd observation under “normal” acquisition rates:
3 frames/min for 1 h or 1 min at maximum speed. Cell retrac-
tion seen with dodecahedron harboring fi ber is a cellular
response to fi ber signaling as proved by fi xed cell controls [
14
].
8. Optional: Before mounting, perform other labeling using
primary antibody (
μ
-clathrin, etc.) and an
appropriate secondary labeled antibody. At the end of the
experiment the nucleus can be stained by 3 min incubation
with Hoechst or propidium iodide as shown in Fig.
4a
.
9. Optional: Other live labeling can be performed at the same
time (GFP-protein as shown in Fig.
4b
, Lysotracker, Hoechst
33342, Organelle-lights, etc.) by following provider instruc-
tions. Carry out the acquisition using suitable channels and
frame rate (maximum speed for short events or 3 frames per
minute for 1 h observation) (
see
Note 7
).
10. “Tip”: We recommend injecting the analyte in the same run-
ning buffer to avoid shifts in the curve at the injection start and
end points. These shifts result from a short delay in time of
α
-actine,
α
-tubuline,
α
