Chemiluminescence nucleic acid detection kit

Example 15

In accordance with the manufacturer's instructions, EMSAs were detected using a chemiluminescent EMSA kit (Thermoscientific, IL, USA) commercially available from Lightshift. Biotin-top and bottom probe parts (5′-CTTCATTTCCCGGAAATCCCTA-Biotin3′, SEQ NO. 11 and 5′-TAGGGATTTCCGGGAAATGAAG-Biotin3′, SEQ NO. 12) of Stat3 probe were annealed and the double-stranded oligonucleotide was end-labelled with biotin. Nuclear extracts from MCF-7 and MDA-MB-231 cells were produced as suggested in the following Reference Document (Choi H S, Hwang C K, Kim C S, Song K Y, Law P Y, Wei L N and Loh H H. Transcriptional regulation of mouse mu opioid receptor gene: Sp3 isoforms (M1, M2) function as repressors in neuronal cells to regulate the mu opioid receptor gene. Mol Pharmacol. 2005; 67(5):1674-1683).

Biotin-labelled DNA probes were cultured together with PAA-treated nuclear proteins in a total volume of 20 μl of EMSA buffer containing 1 μg/μl poly[dI-dC]) for 20 minutes at room temperature. The reaction mixture was subjected to electrophoresis on 4% polyacrylamide nondenaturing gel at 4° C. in the presence of 0.5×TBE (45 mM Tris borate and 1 mM EDTA) and visualized using a chemiluminescence nucleic acid detection kit (Thermoscientific, IL, USA).