Biology Reference
In-Depth Information
11. Fluorescence intensity changes can be fitted to a sum of sig-
moids using Origin, according to the following expression:
=+
−
+
BA
Cx
CD
n
BB
C
−
∑
II A
/
1
1
+
B
+
i
i
−
1
0
1
i
−
1
−
1
−
x
i
=
2
1
exp
1
1
+
exp
i
−
CD
i
11
i
where
A
i
and
B
i
are the lower and upper platform values for
each sigmoid (notice that starting from the second sigmoid,
the lower platform
A
i
is forced to match with the previous
upper platform
B
i
−1
);
C
i
is the transition midpoint; and
D
i
is
the slope. The number of sigmoid curves in the summatory
(up to
n
= 3) is taken as the minimum necessary to fit the
experimental curves, based on best
R
2
values. Higher slope
values indicate high cooperativity effects.
1. Prepare several dilutions of the purified adenovirus sample.
A virus concentration around 1 × 10
11
vp/mL usually gives
good results, but since the spreading of particles on the grid
may vary depending on the hydrophobicity of the carbon sup-
port, it is generally a good idea to try different dilutions every
time, even if using samples with the same concentration.
2. Glow discharge collodion/carbon coated grids. Glow discharge
makes the carbon surface more hydrophilic and facilitates
homogeneous spreading of the particles on the support. Use
glow discharged grids within the next 30 min.
3. Place a piece of Parafilm (about 20 cm long) on the bottom of
a large petri dish and apply pressure to its corners to attach it
to the dish bottom. Place a piece of Whatman paper next to
the dish.
4. Prepare three lines with drops of the samples (first line) (one
drop for each dilution), washing buffer (second line), and
staining agent (third line) on the Parafilm strip as follows:
(a) Sample: one 5 μL drop.
(b) Washing buffer: three 50 μL drops (filtered) (
see
Note 7
).
(c) Staining agent: one 10 μL drop (filtered).
5. Place the grid over the first drop (sample) with the carbon side
facing the drop. Wait for 3-5 min to allow sample adsorption.
6. Blotting: Use high precision tweezers to hold the grid at the
continuous ring of metal at the edge. Carefully, remove the
excess fluid by touching on the edge of the grid with a piece
of Whatman paper, without allowing it to become com-
pletely dry.
3.2 Negative
Staining Transmission
Electron Microscopy
