Biomedical Engineering Reference
In-Depth Information
effi ciency does vary from plate to plate and we have found that (a) a standard
curve is required on each microtiter plate for accuracy and (b) it is essential that
all of the experimental conditions are kept constant (i.e., incubation times). The
standard curve establishes the dynamic range of detection (i.e., the amount of
target protein required to produce linear differences in substrate hydrolysis) and
permits quantitative assessment of target protein concentration in experimental
lysates. The concentration of target protein in mixed protein lysates must fall
within this dynamic range for accurate quantitation.
4. In the most sensitive of assays, detection is achieved by incubation with an
unconjugated detection antibody, a biotinylated secondary antibody, and tertiary
reaction with enzyme-conjugated streptavidin. This method enhances detection
of a single antigen-antibody interaction up to eightfold. If tertiary enhancement
is required, the modifi cations to the protocol outlined in
Subheading 3.3.
should
be made:
1-5. These steps remain unchanged.
6. Amplify the antibody-antigen-antibody complex signal with biotinylated
anti-mouse IgG (1
200,000, Sigma) or appropriate secondary antibody.
Add 50
L per well. Cover with plastic adhesive or wrap and incubate
for 1 h at 37°C.
µ
7. Remove unbound antibody and wash plates as described in
steps 1
and
2
.
8. Detect this complex with extravidin peroxidase (2
µ
g/mL, Sigma). Add
50
L per well. Cover with plastic adhesive or wrap and incubate for 1 h
at 37°C.
µ
9. Remove unbound antibody and wash plates as described in
steps 1
and
2
.
10. Add 75
L per well of TMB substrate and incubate for 10-30 min at room
temperature.
µ
11. Stop reaction by addition of 25
µ
L of 1
M
H
2
SO
4
. The solution will turn
a yellow color.
12. Read the plate on a microplate reader at 450 nm against a reference
wavelength of 650 nm.
References
1. Bruzzone, R., White, T. W., and Paul, D. L. (1996) Connections with connexins:
the molecular basis of direct intercellular signaling.
Eur. J. Biochem.
238,
1-27.
2. Onn, S. P. and Grace, A. A. (1999) Alterations in electrophysiological activity
and dye coupling of striatal spiny and aspiny neurons in dopamine-denervated rat
striatum recorded in vivo.
Synapse
33,
1-15.
3. Onn, S. P. and Grace, A. A. (2000) Amphetamine withdrawal alters bistable states
and cellular coupling in rat prefrontal cortex and nucleus accumbens neurons
recorded in vivo.
J. Neurosci.
20,
2332-2345.
4. Bennett, S. A. L., Arnold, J. M., Chen, J., Stenger, J., Paul, D. L., and Roberts,
D. C. S. (1999) Long-term changes in connexin32 gap junction protein and mRNA
expression following cocaine self-administration in rats.
Eur. J. Neurosci.
11,
3329-3338.
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