By Michael J. May
Focusing on crucial perception into the tools and methods required to dissect the complicated mechanisms of NF-κB activation, rules, and serve as. NF-kappa B: equipment and Protocols guides readers via usual ways to discover NF-κB pathway activation, detection and research of NF-κB signaling, and techniques to review the keep watch over of NF-κB signaling. As a quantity within the hugely winning Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible protocols, and tips about troubleshooting and fending off identified pitfalls.
Comprehensive and authoritative, NF-Kappa B: equipment and Protocols provides a well timed and necessary source for researchers trying to practice experiments geared toward figuring out the position of NF-κB signaling in healthiness and disease.
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Additional resources for NF-kappa B: Methods and Protocols
13. 14. 15. 16. location analysis of mammalian transcription factors. Methods Enzymol 376:304–315 Furey TS (2012) ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat Rev Genet 13:840–852 Martone R, Euskirchen G, Bertone P, Hartman S, Royce TE, Luscombe NM, Rinn JL, Nelson FK, Miller P, Gerstein M, Weissman S, Snyder M (2003) Distribution of NF-kappaB-binding sites across human chromosome 22. Proc Natl Acad Sci U S A 100:12247–12252 Antonaki A, Demetriades C, Polyzos A, Banos A, Vatsellas G, Lavigne MD, Apostolou E, Mantouvalou E, Papadopoulou D, Mosialos G, Thanos D (2011) Genomic analysis reveals a novel nuclear factor-kappaB (NF-kappaB)binding site in Alu-repetitive elements.
Individual primers must be designed to anneal to the same sequence on opposite strands of the plasmid DNA with both primers containing the mutation. Ideally, primers should be between 25 and 45 bases in length with the desired mutation in the middle of the primer. The purity of the primers is critical for high-efficiency mutagenesis. Primers should be purified either by high-performance liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis (PAGE). An A/T to G/C or G/C to A/T substitution of 3–4 bases of the NF-κB binding site should be sufficient to abolish NF-κB binding.
All steps are performed in sterile culture. 2. 7 cells are grown in DMEM containing FCS 10 % (v/v), glutamine, penicillin, and streptomycin. Maintain cells at 37 °C in a humidified environment with 5 % CO2. 3. Cells are passaged by first washing in a pre-warmed (37 °C) medium and detached using a cell scraper. 4. Detachment of cells from tissue culture flask should be visible by eye, but complete detachment should be confirmed using a microscope. 5. 4 cells per well of a 24-well tissue culture plate in a total volume of 500 µl DMEM supplemented with serum, glutamine and antibiotics (see Note 25).