Biomedical Engineering Reference
In-Depth Information
2.4. Western Blotting
1. Polyvinylidene difluoride (PVDF) membrane from Millipore
and 3 MM Chr chromatography paper from Whatman.
2. Activation buffer: 96% (v/v) ethanol.
3. Transfer buffer: 50 mM borate, pH 9.0 and 20% (v/v)
ethanol.
4. Trans-blot SD Semi-dry Electrophoretic Transfer Cell blotting
apparatus (BioRad).
5. Phosphate-buffered saline with Tween (PBS-T): PBS including
0.1% (v/v) Tween 20.
6. Blocking buffer: 5% (w/v) nonfat dry milk in PBS-T.
7. Primary antibodies: mouse monoclonal anti-Hsp70 (Hsp72)
antibody (SPA-810, Stressgen) diluted 1:25,000 in PBS-T;
mouse monoclonal anti-HO1 antibody (OSA-110, Stressgen)
diluted 1:10,000 in PBS-T.
8. Secondary horseradish peroxidase (HRP) conjugated anti-
mouse antibody (P0447, DAKO) diluted 1:25,000 in PBS-T.
9. Enhanced chemiluminescent (ECL Plus
TM
) Western blotting
detection reagent (GE Healthcare) and Phosphor-Imaging
(STORM 840; Molecular Dynamics).
3. Methods
Fibroblasts from one healthy control person are cultured and
used as model for studying stress responses following exposure to
heat shock (1 h at 44°C) and oxidative stress (600 µmol/L H
2
O
2
).
A recovery time of 24 h is used, and the expression of Hsp70 and
HO1 is investigated in cell extracts using specific antibodies as
well as by quantitative PCR. The stress response profiles are com-
pared to fibroblasts which have not been stressed. The profiles
clearly show that fibroblasts respond differently to heat and oxi-
dative stress, suggesting that the expression of Hsp70 and HO1
may be regulated through different mechanisms. The stress treat-
ments performed in this study can be regarded as an “acute”
stress, which activates the appropriate adaptive stress response
systems necessary to alleviate severe cellular damage. Accordingly,
this approach may be useful for studying stress responses in patient
cells, and in addition to Hsp70 and HO1 new potential stress
response components can be investigated and selected as stress
markers for other studies. Fibroblasts could also be exposed to
the stress treatments for prolonged periods of time and combined
with analysis of cell viability it may be possible to deduce if patient
cells exhibited increased sensitivity to stress compared with con-
trol cells.
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