Stress Response Profiles in Human Fibroblasts Exposed to Heat Shock or Oxidative Stress - Protein Misfolding and Cellular Stress in Disease and Aging - page 161

Fig. 1. Principle of the TaqMan chemistry ® . The TaqMan ® gene expression assay includes two unlabelled primers for
amplification and one TaqMan ® probe for detecting the sequence of interest. The probe contains a reporter dye (R) linked
to the 5 ′ -end and a quencher (Q) at the 3 ′ -end, which anneals specifically to the target sequence between the two primer
sites. When the probe is intact, the reporter fluorescence will be suppressed by the quencher. During the PCR, 5 ′ -3 ′
exonuclease activity of the AmpliTaq Gold ® DNA polymerase cleaves the probe and separates the reporter from the
quencher, resulting in increased fluorescence of the reporter. The probe fragments are then displaced from the target,
and elongation of the strand continues. Accumulation of PCR products can be detected directly by monitoring the increase
in fluorescence of the reporter dye using an ABI PRISM ® 7000 Sequence Detection System from Applied Biosystems.
3. Determine protein concentration of the supernatant fraction
using the Bradford protein assay.
4. Dilute protein extracts to appropriate concentrations in 1:6
volumes of SDS sample buffer, mix, and boil 5 min in a heat-
ing block (95°C).
5. Centrifuge briefly and keep the samples at room temperature.
6. Separate the proteins by 12.5% SDS-polyacrylamide gel elec-
trophoresis (PAGE) in cold 1× SDS running buffer. Use
10 µg protein for HO1 blots and 2 µg for Hsp70 blots.
Include one well for prestained molecular weight marker
(3 µl).
7. Run the gel at 200V for 60-70 min or until the dye front is
reaching the bottom of the gel (4°C).