Biomedical Engineering Reference
In-Depth Information
Table 1
Microplate Layout for Experimental Detection of Cx32 in Medulla,
Parietal/ Temporal Cortex, Cerebellum, Thalamus, Dorsal Striatum,
Nucleus Accumbens, and Globus Pallidus/Ventral Lateral Striatum
in Drug-Naive (Control) Animals and in Chronic Cocaine
Self-Administrators After 2, 7, and 21 d of Cocaine Withdrawal
1
2
3
4
5
6
7
8
9
10
11 12
A
Blank
0 µg
M
M
CB
CB
DS
DS
VS
VS
protein
Control Control Control Control Control Control Control Control
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
B
Blank
0 µg
M
M
CB
CB
DS
DS
VS
VS
protein
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
C
Blank
0 µg
M
M
CB
CB
DS
DS
VS
VS
protein
7 d
7 d
7 d
7 d
7 d
7 d
7 d
7 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
D
Blank
0 µg
M
M
CB
CB
DS
DS
VS
VS
protein
21 d
21 d
21 d
21 d
21 d
21 d
21 d
21 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
E
Blank
0 µg
Cx
Cx
T
T
NA
NA
protein Control Control Control Control Control Control
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
F
Blank
0 µg
Cx
Cx
T
T
NA
NA
protein
2 d
2 d
2 d
2 d
2 d
2 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
G
Blank
0 µg
Cx
Cx
T
T
NA
NA
protein
7 d
7 d
7 d
7 d
7 d
7 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
H
Blank
0 µg
Cx
Cx
T
T
NA
NA
protein
21 d
21 d
21 d
21 d
21 d
21 d
20 µg
20 µg
20 µg
20 µg
20 µg
20 µg
Layout of a 96-well plate and loading of experimental samples as described in
Subheading
3.2.2.
is illustrated. Blank: Wells were coated with R5 capture antibody and blocked with blocking
solution. In all subsequent steps, wells were incubated with the appropriate buffers in the absence
of protein lysate or antibodies. These values are set to 0 by the microplate reader during the run.
0 µg protein: Wells were treated exactly as described for experimental samples except protein was
not included in the 40 µL of RIPA and 10 µL of coating buffer (
Subheading 3.2.2.,
step 1
).
This control determines the lower limit of assay sensitivity and establishes nonspecifi c binding
of all reagents in the absence of target protein. Experimental values that fall below this value
cannot be interpreted. If this absorbance is high, increase the number of washes. M, Medulla;
Cx, parietal-temporal cortex; CB, cerebellum; T, thalamus; DS, dorsal striatum; NA, nucleus
accumbens; VS, ventral striatum (not including the nucleus accumbens). Control: Samples obtained
for drug-naive animals. 2 d: Protein extraction performed 2 d after the last cocaine injection. 7 d:
Protein extraction performed 7 d after the last cocaine injection. 21 d: Protein extraction performed
21 d after the last cocaine injection. Experimental samples represent protein lysates extracted from
tissue pooled from two animals.
Search WWH ::
Custom Search