Biology Reference
In-Depth Information
DKZeo 60-80 % confl uent overnight under agitation. 36 h
after, the same procedure is applied to a new 10 cm Ø and
subsequently 3× and then 10 × 10 cm Ø before applying super-
natants to 30-50 15 cm Ø dishes for the purifi cation.
3. Cracked cells (resuspended in 40 mL of culturing medium) are
then loaded on top of a step gradient (six tubes consisting of
2 mL 1.25 g/mL CsCl + 2 mL 1.45 g/mL CsCl) and ultracen-
trifuged for 1.5 h at 218,000 ×
g
at 18 °C. The opaque band at
the interface is taken with a syringe (~ 4 mL for the 6 tubes)
and further resuspended in 8-9 mL of 1.32 g/mL CsCl (iso-
pycnic gradient). Following 18 h of ultracentrifugation at
218,000 ×
g
at 18 °C, the bottom band is collected in 1 mL
and dialyzed three times against PBS (with the last dialysis in
PBS-10 % glycerol).
4. Multiplicity of infection (physical particles (pp)/mL) is deter-
mined by nanodrop. Infectivity (infectious particle/mL) is
tested in DKZEo.
5. For labeling, 2 × 10
12
pp are diluted in 100
μ
L of PBS and
added to 100
L of PBS
0.1 M HCO
3
for 30 min at room temperature (RT). To remove
unconjugated dye, the solution is then dialyzed overnight
against PBS and for 3 h against PBS-10 % glycerol (
see
Note 2
).
Infectivity is tested in DKZEo.
μ
L of Cy3 mono-reactive dye and 300
μ
1.
Escherichia coli
strain BL21 is transformed with a CAdV-2
Fiber knob plasmid containing an N-terminal His
6
tag and a
TEV protease cleavage site [
14
]. After cells growth in LB6 amp
at 37 °C, protein expression is induced by IPTG (1 mM) for
1 h. The culture is then centrifuged at 2,000 ×
g
.
2. The purifi cation is performed with the kit Protino Ni-TED
1000. Briefl y, cells are resuspended in lysis buffer and lysed by
sonication. The cell lysate is centrifuged at 10,000 ×
g
for
30 min at 4 °C, and the supernatant is loaded on a Ni
2+
-TED
(tris-carboxymethyl ethylene diamine) column. His-Tag
proteins bound to the resin are then washed and eluted.
3. To separate the His tag from the fi ber knob (FK), FK are
incubated for 1 h 30 with His-tagged TEV protease at 30 °C.
Uncleaved protein and TEV protease are removed by binding
to a Ni
2+
-agarose beads.
4. A Cy5 mono-reactive dye pack is used to label CAdV-2 FK.
200
3.2 Production and
Labeling of CAdV-2 FK
μ
g of FK (200
μ
L) is mixed with 50
μ
L of the fl uorescent
dye (one vial resuspended in 500
L of
PBS 0.1 M HCO
3
and incubated for 30-45 min at RT. Labeled
proteins are eluted with PBS on a NAP5 column. The fi nal
dye/protein ratio (~2.4) is analyzed by spectrophotometer
using a NanoDrop ND-100.
μ
L of H
2
O) and 25
μ
