By Hans-Joachim Anders, Adriana Migliorini
Innate DNA and RNA reputation: technique and Protocols provides established experimental ideas to dissect nucleic acid sensing in-vitro and in-vivo assets. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, with ease reproducible laboratory protocols and pointers on troubleshooting and averting identified pitfalls.
Authoritative and practical, Innate DNA and RNA popularity: procedure and Protocols presents a source for immunologists, molecular biologists, virologists, microbiologists and researchers learning how the innate immune approach handles nucleic acids from endogenous or overseas sources.
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Extra resources for Innate DNA and RNA Recognition: Methods and Protocols
All local template sequences are then combined to generate a multiple sequence alignment with the full-length target sequence. In this way, a high-quality model can be created, even if no adequate full-length template is available (see Note 3). 1 Model Construction 1. TollML database: a database of sequence motifs of TLRs . TollML includes all known TLR protein sequences extracted from the NCBI protein database . Each sequence was semi-automatically partitioned into four parts: signal peptide, 48 Jing Gong and Tiandi Wei ectodomain, transmembrane domain, and TIR domain.
Spin down the samples containing the pre clearing beads at 4 °C at 1,000 × g and transfer the supernatant of the pre clearing step onto the anti-FLAG coupled beads or beads coated with unspecific IgGs. 13. Incubate for 2 h at 4 °C on a tube roller as described for the pre clearing beads. 14. Spin down the beads at 4 °C at 1,000 × g. 5 ml Eppendorf tubes. Take 20 μl of the supernatant for western blot analysis and dilute in 2× Laemmli buffer. Freeze at −20 °C. These samples will from now on be referred to as “supernatant”.
Human IP-10 ELISA kit. 22. Vesicular stomatitis virus (Indiana strain). 40 3 Andreas Schmidt et al. 1 Purification of RIG-I-Bound RNAs from VSV-Infected Cells Hek293 FLAG-RIG-I cells were cultivated in medium I. For the control condition lacking the over-expressed protein Hek293 FlpIn cells were cultivated in medium II. We typically run the conditions: ● Virus-infected and FLAG-RIG-I-induced cells immunoprecipitated with anti-FLAG-coupled agarose. ● Virus-infected and FLAG-RIG-I-induced cells immunoprecipitated with IgG-coupled agarose.
Innate DNA and RNA Recognition: Methods and Protocols by Hans-Joachim Anders, Adriana Migliorini