Biotin end-labeled oligonucleotides were conjugated to Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, 11205D) for 30 min at room temperature according to the manufacturer’s protocol. Conjugated beads were incubated for 2 hours at 4°C with cleared lysates from confluent cells transfected with the appropriate overexpression plasmids. The beads were then washed four times with wash buffer, and the bound proteins were eluted by 95% formamide, 10 mM EDTA, and free biotin at 65°C for 10 min and then analyzed by Western blotting. The oligonucleotides used are as follows: hIL6 3′UTR, 5′-biotin-UAUUUUAAUUAUUUUUAAUUUA-3′; 2×GAS, 5′-biotin-CGTTTCCCCGAAATTGACGGATTTCCCCGAAAC-3′ (forward) and 5′-GTTTCGGGGAAATCCGTCAATTTCGGGGAAACG-3′ (reverse); nonGAS, 5′-biotin-CGTTTACCCCAAATTGACGGATTTACCCCAAC-3′ (forward) and 5′-GTTGGGGTAAATCCGTCAATTTGGGGTAAACG-3′ (reverse); newMOTIF, 5′-biotin-CGTTTTTGAGGCAATTGACGGATTTTTGAGGCAAC-3′ (forward) and 5′-GTTGCCTCAAAAATCCGTCAATTGCCTCAAAAACG-3′ (reverse). All oligos were purchased from Thermo Fisher Scientific.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.