Biochemical Assessments of Cerebral Vasospasm: Measurement of cGMP, PKC, and PTK in Cerebral Arteries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 517

4.2. Solutions

All concentrations are expressed in mM except when specifi ed.
Homogenization buffer . 50 Tris, 5 EDTA, 10 EGTA, 1 phenylm-
ethylsulfoxide, 10 benzamidine, 25 mg/mL leupeptin, and 250
sucrose.
×2 modifi ed Laemmli sample buffer . 125 Tris, 2% sodium dodecyl
sulfate (SDS), 20% glycerol, and 16 dithiothreitol (pH 6.8).
Electrophoresis buffer . 25 Tris, 192 glycine, 0.10% SDS (pH 8.4).
Transfer buffer . 25 Tris, 192 glycine, and 20% methanol (pH
8.3).
Tris-buffered saline (TBS) . 10 Tris, 150 NaCl (pH 7.5).

Canine basilar arteries are obtained as described in Sect. 1.1 . As a
positive control for these experiments, a small piece of rat brain
and canine brain are also collected. Tissues are placed into 1.5-mL
plastic tubes and homogenized with a polytron in ice-cold homog-
enization buffer (400 mL). After incubation for 30 min on ice, an
equal volume of ×2 modifi ed Laemmli sample buffer ( 21 ) is added.
Samples are boiled to denature proteins for 5 min and then cooled
in ice-cold water. Samples are stored at −20°C until used.

4.3. Sample
Preparation

4.4. SDS-PAGE/
Western Blotting

Proteins are separated by molecular weight using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The acryl-
amide concentrations in gels are selected based on the molecular
weight of objective protein(s). For example, a high concentration
of acrylamide (~15%) is recommended for small molecular weight
proteins (~20 kDa), and low concentration (~7.5%) is used for
large proteins (>150 kDa). As the molecular weight of PKC iso-
forms is 65-120 kDa, 10% acrylamide gel is used here for separa-
tion (separate gel; 10% acrylamide (29:1 acrylamide/bis-acrylamide),
375 mM Tris-HCl (pH 8.8), 0.1% SDS, 0.05% N , N , N ¢, N ¢-
tetramethylethylenediamine (TEMED), 16.9 mM ammonium per-
sulfate (APS), also ready-to-use precast gels are commercially
available). After adding bromophenol blue (fi nal concentration
0.1%), samples are loaded on a stacking gel (4% acrylamide,
125 mM Tris-HCl (pH 6.8), 0.1% SDS, 0.1% TEMED, 22 mM
APS), which is layered on a separate gel. Molecular markers are
also applied to the gel to estimate the molecular size of obtained
signal(s). Samples are initially run through a stacking gel at 20 mA
for ~15 min. Following this period, the samples are concentrated
at the edge of the separation gel and appear as a thin blue line. At
this time, the current is increased to 40 mA and proteins are sepa-
rated until the thin blue line approaches the bottom of the separa-
tion gel. The blue line should not run off the gel. Some commercially
available gels consist of single layer with a single concentration of
acrylamide gel, i.e., only separation gel. In this case, current is ini-
tially set at 40 mA. If running two gels at the same time, twice as
much current is needed.