Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 633

1.1. Sample
Preparation

1. Rats are sacrifi ced under deep anesthesia at different time
points after intracerebral hemorrhage (ICH) induction or an
injection of saline.
2. Following intracardiac perfusion with phosphate-buffered
saline (PBS), the brains are removed, and a 3-mm thick coro-
nal brain slice is cut 4 mm from the frontal pole.
3. The ipsilateral and contralateral basal ganglia samples are dis-
sected from the slice.
4. The brain samples are then immersed into 500 mL of sample
buffer and sonicated on ice.
5. Protein concentration is determined by Bio-Rad protein assay
kit.

1. A 12.5% polyacrylamide separating gel (MW of HO-1: 32 kDa,
Table 1 ) is prepared. After the separating gel has set, cast a
4.5% polyacrylamide stacking gel on top with a comb to form
sample wells (Fig. 1b ).
2. Prepare the brain samples for loading onto the gel (50 mg pro-
tein in 20 mL total volume). Boil for 5 min at 95°C, cool, and
load into the sample wells on the gel along with a prestained
MW marker (Fig. 1c ).
3. Carry out electrophoresis at 30 mA constant current until the
sample has traversed the stacking gel. During this time, the
samples “stack,” becoming concentrated into a very tight
band, affording maximal resolution and separation of the poly-
peptides in the separating gel. Then continue electrophoresis
until the bromphenol blue dye front is approximately 1 cm
from the end of the gel.
4. Remove the gel assembly from the vertical gel apparatus, sepa-
rate the glass plates, and cut off the stacking gel with a sharp
scalpel blade.

1.2. SDS-PAGE
Electrophoresis

1. For each gel, cut a piece of nitrocellulose and two pieces of
Whatman 3 M fi lter paper to 5.5 × 8.5 cm.
2. Open the plastic holder for the gel/membrane “sandwich” in
a basin containing suffi cient transfer buffer to cover it. Place a
nylon pad fl at into the holder, followed by a piece of fi lter
paper, gel, and then the nitrocellulose. Roll over the nitrocel-
lulose with a glass rod to remove any air bubbles between the
gel and the membrane.
3. Lay another piece of fi lter paper and nylon pad on top, and
carefully close the blotting clamp. Place the clamp in the blot-
ting apparatus.
4. Fill the tank with the transfer buffer, suffi cient to cover the
gel/membrane “sandwich.” Carry out the transfer at a constant
current of 30 mA overnight at 4°C.

1.3. Assembly
of the Gel/Membrane
“Sandwich”