Bound proteins were eluted in 30 µl of 50 mM Tris-HCl (pH 6.8) containing 100 mM glutathione, were resolved by SDS-polyacrylamide gel electrophoresis, and were visualized and quantified by PhosphorImager analysis.
5 pmol of labeled probes were incubated with 50 pmol of purified recombinant GST-AP-2γ protein for 20 min at 37°C in 15 μl of reaction buffer: 10 mM HEPES (pH 8.0), 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 100 μg/ml BSA, 0.25 mM DTT, and 10% glycerol; then were resolved on 6% non-denaturation polyacrylamide gels.
The chemical crosslinking reaction was stopped by adding 50 mM Glycine and the samples were resolved by SDS-PAGE [15].
At 50 mm working distance, 5.04 lp/mm were resolved.
Twenty micrograms of lysates, reduced with 100 mM DTT or left untreated, were resolved by Tris glycine SDS PAGE and transferred to PolyScreen PVDF Transfer Membrane (Perkin Elmer) by submarine transfer.
Whole cell lystaes (75 μg) were prepared in a lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40), and were resolved by a 10% sodium dodecyl sulfate (SDS -polyacrylamide gel (SDS -polyacrylamide by beingelransferred to a PVDF membrane (Millipore, USA).
The pellets were resolved in a hypertonic buffer (20 mM HEPES, 50 mM KCl, 300 mM NaCl, 1 mM PMSF, 1 mM DTT, and protease inhibitors) to obtain nuclear extracts.
Powdered samples were resolved in 10 mM HCl, mixed in Tissue Lyser II at 25 hertz for 1 min, and centrifuged at 20,500 g for 15 min at room temperature.
All PCR products were resolved on 1 mm, 5% acrylamide gels, stained with ethidium bromide, and visualised by transillumination.
Normal nucleotides were resolved in 180 mM sodium phosphate, pH 6.0, by one-directional PEI-cellulose TLC.
