Biomedical Engineering Reference
In-Depth Information
3.5. Western Blotting
1. Transfer the separated proteins onto a PVDF membrane for
45 min at 220 mA using a Trans-blot SD Semi-dry
Electrophoretic Transfer Cell blot apparatus from BioRad.
Activate the PVDF membrane in 96% (v/v) ethanol
(5-40 min) and wet gel, membrane and 3 MM chromatogra-
phy paper in transfer buffer prior to blotting.
2. Once the transfer is completed, rinse the PVDF membrane in
PBS-T and incubate 1 h in 25 ml blocking buffer.
3. Rinse twice in PBS-T (2 min) prior to addition of 25 ml of a
1:25,000 dilution of the anti-Hsp70 antibody in PBS-T or
25 ml anti-HO1 antibody diluted 1:10,000 in PBS-T.
Incubate overnight at room temperature on a rocking plat-
form. Use a sealed container!
4. Discard primary antibody and rinse the blot twice in PBS-T
(2 min) followed by a 15-min wash and then three times
5-min wash in PBS-T.
5. Add 25 ml 1:25,000 secondary antibody solution in PBS-T
and incubate 1 h at room temperature on a rocking
platform.
6. Discard secondary antibody and rinse the blot twice in PBS-T
(2 min) followed by a 15-min wash and then three times
5-min wash in PBS-T.
7. During the final wash, warm aliquots of ECL reagents A and
B to room temperature. Once the final wash is removed from
the blot, ECL reagents are mixed together, immediately
added to the blot and rotated by hand to ensure even cover-
age. Use a lightproof container!
8. Incubate for 5 min and seal the blot in a plastic bag.
9. Use Phosphor Imaging (STORM840; Amersham Biosciences)
for visualization and the Image Quant
®
software for quantifi-
cation. See Hsp70 and HO1 protein expression profiles in
Fig.
2
.
4. Notes
1. The phenyl-methyl sulfonyl fluoride (PMSF) should be added
to the lysis buffer immediately prior to use. As an alternative,
leupeptin, aprotinin, and PMSF could be replaced by a protease
inhibitor cocktail tablet, for example, Complete, Mini, EDTA-
free from Roche Applied Science (Cat # 11836170001).
2. If you use a heat incubator without CO
2
for the heat shock
treatment we recommend you to change the growth medium
on the day before the stress treatment so that the medium is
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