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14. Discard DTT solution and replace with equilibration buffer
plus 4.8% (w/v) iodoacetamide. Incubate for another 15 min.
15. Briefl y wash the strips with ddH
2
O, then place them on top of
the gels and add 3 mL of agarose sealing solution. Ensure that
there are no bubbles between the IPG strips and the gel sur-
face. Let the agarose set.
16. Run the second dimension. We run the gels on an Ettan
DALT
six
Electrophoresis Systems (GE Healthcare) at 2 W/gel
for 15 min, 2.5 W/gel for 30 min before the power is increased
to 30 W/gel. The run is continued until the bromophenol
blue dye front migrates off the lower end of the gels.
4. Notes
1. If the tissue samples remain for a prolonged period of time in
the hypertonic solution, cell membranes may collapse as a
result of the continuous loss of water from the cells, thereby
causing loss of cellular proteins in the buffer. In our experi-
ence, vascular samples, especially arteries, which are rich in
ECM, withstand 4 h of incubation with 0.5 M NaCl. In con-
trast, the incubation period should be restricted to 1 h for car-
diac tissues. Also, buffer-to-tissue weight ratio is crucial; if the
ratio is too low, then the buffer will be quickly saturated with
extracted proteins, and the effectiveness of the extraction will
be reduced. The extracted protein concentration is diffi cult to
predict, but in practice, a 10:1 ratio provides good extraction
effi ciency without excessive dilution of the sample.
2. The optimal incubation time is strongly dependent on the cel-
lularity of the tissue. In our experience, a 4-h incubation is
suffi cient for vascular tissues, but 16 h are required for retrieval
of cellular proteins in cardiac tissues. Yet, some myofi lament
proteins will still remain, and we have used other protocols to
study this particular cardiac subproteome (
8
).
3. Notes on collagen solubilization: Interstitial collagen is very
diffi cult to solubilize. Acidity is essential together with the
action of pepsin, which cleaves collagen at the telopeptide
region, thereby increasing its extraction effi ciency (
15
).
4. Removal of the chaotropic agent is essential for deglycosyla-
tion. Its denaturing properties would inhibit enzymatic activity.
Also, the ionic strength of the buffer would negatively affect
separation by gel electrophoresis. Guanidine is readily soluble
in ethanol, which will also precipitate the extracted proteins.
Most of the heavily glycosylated ECM proteins are in the 4 M
guanidine extracts.
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