Biology Reference
In-Depth Information
3. Follow preparation of antibody array membranes, all solutions,
incubation times with buffers, and all washing procedures as
described in Subheading
3.1
from
steps 4
-
25
.
4. Expose membranes to the fi lm for 15 s, 30 s, 1 min, 3 min,
5 min, and 10 min.
5. Select a set of fi lms that were exposes to the membrane for
exactly the same period of time and show the most signifi cant
difference in the dynamic range of a histogram density units,
and plot a calibration curve for each particular cytokine and
chemokine (
see
Notes 2
and
3
). An example of the results pro-
duced for calibrating concentrations of MIP-2, MCP-1, and
KC is shown in Fig.
1
.
6. Follow procedures in an exactly the same way while processing
the tissue samples, collected after Ad injection, and expose the
membranes to the fi lm for exactly the same time as calibration
curve membranes.
Fig. 1
Detection of increasing concentrations of recombinant purifi ed murine IL-1
and MIP-2 chemokines,
spiked with 50 mg of spleen sample, using (
a
) ProteomeProfi ler™ antibody arrays, and (
b
) calibration curves
derived from quantifying pixel density of indicated spots on the membrane for IL-1
α
and MIP-2 using histo-
gram tool of the Adobe Photoshop software. Representative blots, exposed for 15 and 60 s to corresponding
membranes are shown
α
