PCR products were concentrated to 20 μl and resolved by electrophoresis on 1.4% agarose gels in Tris-borate-EDTA (TBE) buffer, and were detected by ethidium bromide staining [ 24].
In preliminary experiments, PCR products (10-μL aliquots) were resolved by electrophoresis on 3.5% MetaPhor agarose (FMC Bio-Products, Rockland, ME, USA) gels in Tris-borate-EDTA (TBE) buffer, and were detected by ethidium bromide staining.
Final extension was carried out at 72°C for 10 min. Amplified PCR products were resolved on 1.5% agarose gels with 1× TAE buffer (pH 8.0) and were detected under UV light after ethidium bromide staining.
Real-time PCR (ABI7900 Fast Real-time PCR system; Applied Biosystems, Foster City, USA) was performed using the following program: 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 s, 58°C 30 s, and 60°C for 30 s. Real-time PCR products were also resolved on 2% agarose gels with 1× TAE buffer (pH 8.0) and were detected under UV light after ethidium bromide staining.
The bound proteins were eluted, denatured in sample buffer, separated by 4 15% SDS-PAGE and were detected by Western blot analysis using anti-Ku70 antibodies.
After incubation for 1 hour at room temperature with an HRP-conjugated anti-mouse secondary antibody (1∶5000 in blocking buffer), signal was detected using ECL femto reagent (Thermo Scientific, Rockford, USA) and visualized with a BioRad ChemiDoc imaging station and glycotpyes were evaluated using published protocols [41].
Following several more washes in wash buffer, fluorescence was detected using a Leica DM SP2 confocal microscope (Leica, Wetzlar, Germany) and images were assembled using Adobe Photoshop CS4 (Adobe, San Jose, CA).
A strong negative buffer effect was detected when using 50 mM Na2HPO4/KH2PO4 (pH 5.0 to 7.0).
Without treatment of samples with dissociation buffer, MSP1 was detected in 5 of 13 cord blood plasma samples (Figure 2B); following treatment with dissociation buffer, MSP1 detection increased to 12 of 13 cord samples (Figure 2B).
No fluorescence over that of phosphate buffer saline was detected in cells grown in noninducing medium.
