A detailed protocol for DNA binding assay was previously described41 . Briefly, cells treated for 48 hrs with compounds were lysed in 1% NP40/phosphate buffered saline/protease inhibitors and cleared using a microcentrifuge. The lysate was then incubated with 0.5 µM of double-strand biotinylated oligonucleotides and 40 µl streptavidin-agarose bead suspension (Thermo Scientific, 20349) for 2 hrs. The streptavidin agarose beads are washed (3X) with 1% NP40/phosphate buffered saline/protease inhibitors and eluted with 2X Laemmli sample buffer. The supernatants are separated on SDS-PAGE and analyzed using western blotting. Antibodies were purchased from the following vendors: GLI1 (Cell Signaling Technology, #2534) and actin (SIGMA, #A5441).
Streptavidin agarose bead suspension
Manufactured by Thermo Fisher Scientific
Streptavidin-agarose bead suspension is a laboratory reagent consisting of streptavidin, a protein with a high affinity for biotin, immobilized on agarose beads. The beads can be used to capture and isolate biotinylated molecules in various applications.
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Lab products found in correlation
2 protocols using streptavidin agarose bead suspension
1
DNA Binding Assay for Protein Analysis
2
Colorimetric Detection of HRP-Oligonucleotide Conjugates
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HRP-oligonucleotide conjugates for immobilization were prepared as previously described18 (link) using the oligonucleotides shown in Table 1 . The conjugates were immobilized through biotin-streptavidin interactions on the surface of either streptavidin-precoated 96-microwell plates, or the streptavidin agarose bead suspension (both from Thermo Fisher Scientific). HRP was released from the surface attachment as the result of REase cleavage of probes and linkers. Reaction mixtures containing released HRP were transferred to wells of a 96-well ELISA microplate (Thermo Fisher Scientific). Then 100 μL of the BioFX TMB One Component HRP Microwell Substrate (SurModics, Eden Prairie, MN) was added into each well. The HRP-generated signal was quantified after 2–5 min incubation at room temperature by the blue color formation that was measured colorimetrically at the wavelength of 655 nM (OD655), using an iMark Microplate Reader (Bio-Rad).
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