Biology Reference
In-Depth Information
6. Organelles or cytosqueletton detection (Phalloidine
Rhodamine: Sigma; Lysotracker: Invitrogen; Organelle-Lights
Rab5: Invitrogen).
7. Glass coverslip of 0.17 mm for fi xed cells and 0.17 mm Iwaki
dishes with glass bottom for live imaging.
8. Thermostated inverted Olympus microscope IX81 or similar.
1. BIAcore 3000 (GE-Healthcare).
2. CM4 or CM5 censorships (GE-Healthcare).
3. HBS buffer: 20 mM HEPES, pH 7.4, 150 mM NaCl.
4. EDC-NHS Amine coupling Kit (GE-Healthcare).
5. Streptavidin.
6. Biotinylated Heparin or other lab-made biotinylated ligand.
7. Desmoglein.
8. Regeneration solutions: 0.05 % SDS in water or 20 mM HCl
in water.
2.4 Surface
Plasmon Resonance
3
Methods
Adenovirus dodecahedron can be considered an adenoviral mimic.
This is particularly true for receptor interaction studies such as
penton base/integrins or fi ber/desmoglein [
6
,
7
], but neverthe-
less one must keep in mind that some different features exist.
Among them, interaction with HSPGs has never been reported for
the whole Ad3, whereas Ad-Dd interacts at high affi nity with mol-
ecules of this family [
5
]. In addition, the role played by the hexon
in binding coagulation factors has recently been emphasized, but
obviously Ad-Dd behaves differently and can only be used as a
negative control for studies in this fi eld. The main advantages of
the dodecahedron over isolated capsomers (i.e., pentons, fi bers) lie
both within its multivalency and its spatial constellation [
14
].
These properties are especially appreciated for surface plasmon
resonance studies as the higher molecular weight of VLPs is an
advantage in terms of signal and binding avidity can be investi-
gated. In this chapter, purifi cation of Ad-Dd is described, and
methods enabling its detection are reported. Applications of these
tools from a molecular level to a cellular scale are shown.
1. High Five Cells are cultured in a 27 °C thermostatic incubator
or when available (
see
Fig.
1
) in a 27 °C room under gentle
shaking on the Certomat.
2. Cells are regularly counted using a Neubauer counting cell by
adding an equal volume of trypan blue to an aliquot of the cell
culture taken under sterile conditions. The cells are left to
3.1 Production:
Cell Culture and
Baculovirus Infection
