EMSAs were conducted as described (Lee and Gralla, 2001 ; Zhao et al., 2005 ). Briefly, DNA fragments were amplified using the primer pairs shown in Table S1 . PCR amplicons were gel purified with a QIAquick Gel Extraction Kit (Qiagen), and the purified products were labelled with the Pierce™ biotin 3′ end DNA labelling kit (Thermo Fisher Scientific, Rockford, IL). The binding reaction was performed with the non‐specific competitor DNA (poly dI‐dC) and NP‐40 in buffer containing 10 mM HEPES (pH 7.3), 20 mM KCl, 1 mM MgCl2 and 5% glycerol at 25°C for 2 h. The final mixtures were run on a 6% DNA retardation gel (Invitrogen), transferred to a nylon membrane and UV cross‐linked. Chemiluminescence was performed with the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.
Pierce biotin 3 end dna labelling kit
Manufactured by Thermo Fisher Scientific
The Pierce™ biotin 3′ end DNA labelling kit is designed for the labelling of DNA fragments with biotin at the 3′ end. The kit provides the necessary reagents and instructions to perform the labelling reaction.
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2 protocols using pierce biotin 3 end dna labelling kit
1
Electrophoretic Mobility Shift Assay Protocol
2
AlphaScreen Analysis of Nur77 and AIM2 Binding
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Purified proteins and biotinylated LPS or DNA were analyzed with an AlphaScreen assay by incubation at various concentrations according to the manufacturer's instructions (PerkinElmer). 20 nM recombinant Nur77, Nur77 lacking amino acids 524-547 or control GST protein were incubated with biotin-LPS of the indicated concentrations, otherwise 20 nM recombinant Nur77 or AIM2 protein were incubated with biotin-conjugated mitochondrial DNA of the indicated concentrations, followed by an AlphaScreen assay. BMDM mitochondrial DNA was linearized by SphI, followed by an addition of biotin to the 3' end using a Pierce Biotin 3' End DNA Labelling Kit (ThermoFisher Scientific).
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