A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro
| Autor: | Holleufer, Andreas |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Zdroj: | Holleufer, A 2018, ' A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro ', Bio-protocol, vol. 8, no. 20 . https://doi.org/10.21769/BioProtoc.3055 |
| Popis: | Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that sensesdouble stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferongenes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruitsTANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatoryfactor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNAcomplex catalyzes the synthesis of 2’3’-cGAMP and the purpose of the protocol presented here is tomeasure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed toelucidate the relationship between dsDNA length and the level of cGAS activity. The method involvesan in vitro reaction with low concentrations of cGAS and dsDNA followed by quantification of the reactionproduct using anion exchange chromatography. The low concentrations of cGAS and dsDNA and thehigh sensitivity of this assay is a key advantage when comparing different DNA fragments’ ability toactivate cGAS. |
| Databáze: | OpenAIRE |
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