Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 635

1.4. Development
of the Western Blot

1. Carefully remove the nylon pad and top fi lter paper.
2. Cut away any excess nitrocellulose from around the gel with a
pair of scissors.
3. Place the nitrocellulose membrane in a suitable plastic con-
tainer and stain with Ponceau Red solution for 5 min with
gentle agitation (Fig. 1d ).
4. Block the membrane with PBS, pH 7.4, with 0.1% Tween
(PBST) containing 5% nonfat dried milk for 1-2 h. The Ponceau
Red stain will be washed off the membrane during the blocking
step. All subsequent reactions/washings are carried out on a
shaker at room temperature.
5. Wash with PBST (3× 5 min) before incubating with the fi rst
antibody (polyclonal rabbit anti-rat HO-1, 1:2,500 dilution,
StressGen, Victoria, Canada) for 1-2 h at room temperature.
6. After washing with PBST (3× 5 min), place the membrane in the
HRP-conjugated secondary antibody solution (goat anti-rabbit
IgG, 1:2,500 dilution, BioRad) for 1 h at room temperature.
7. After washing with PBST (3× 5 min), add the ECL mix (1:1
mix of Amersham reagents 1 and 2) for 1 min.
8. Dry out the excess liquid, wrap the membrane, and immediately
expose to Kodak X-OMAT fi lm for 30-60 s.
9. The relative densities of the bands are analyzed using NIH
Image J (Fig. 1e ).

1.5. Stripping Blots

1. Most antibodies should be removed by SDS. Wash blot well
with PBST. Incubate and agitate the membrane in stripping
buffer (100 mM b-mercapto-ethanol, 2% SDS, 62.5 mM Tris,
pH 6.8) for 30 min at 50°C in a closed container.
2. Wash several times with PBST. The membranes are ready to
reuse and can be reprobed with antibodies following the block-
ing step.

1. The concentration of acrylamide depends on the molecular
mass of the polypeptide to be identifi ed. As an approximate
guide, for polypeptides of >100 kDa, a separating gel of 5-7%
acrylamide should be employed. For polypeptides between 50
and 100 kDa, 7-9% and for <50 kDa, the acrylamide concen-
tration in the separating gel should be 12-15% (Table 1 ).
Acrylamide and bis -acrylamide are cumulative neurotoxins.
Care should be exercised in their handling.
2. It is advisable to run protein molecular weight standards in one
of the electrophoresis lanes (Fig. 1d ).
3. The dilution of the primary antibody is dependent on the
nature of the antibody (i.e., monoclonal or polyclonal) and
should be determined empirically.

1.6. Notes