NF-kappa B: Methods and Protocols (Methods in Molecular by Michael J. May

By Michael J. May

Focusing on crucial perception into the equipment and methods required to dissect the advanced mechanisms of NF-κB activation, legislation, and function. NF-kappa B: tools and Protocols guides readers via common techniques to become aware of NF-κB pathway activation, detection and research of NF-κB signaling, and techniques to check the keep watch over of NF-κB signaling. As a quantity within the hugely winning Methods in Molecular Biology series, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible protocols, and pointers on troubleshooting and heading off recognized pitfalls.

Comprehensive and authoritative, NF-Kappa B: tools and Protocols provides a well timed and beneficial source for researchers trying to practice experiments aimed toward figuring out the position of NF-κB signaling in future health and disease.

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Incubation temperature is dependent on the restriction enzymes used. 18. Run undigested plasmid DNA on the same gel as a control for restriction enzyme activity. 19. Ligation reactions are generally carried out with an excess of insert DNA. A 3:1 molar ratio of insert to vector is commonly used, but this ratio can be varied from 10:1 to 1:1. 20. Include a negative control ligation in which the insert is replaced by water. This will determine the amount of background colonies formed as a result of incomplete vector digestion or vector religation.

In this article, we present a detailed protocol for the quantification of in vivo ubiquitination and turnover of IκB-α in response to a variety of cellular stimuli. Key words NF-κB, RelA, IκB, Immunoprecipitation, Ubiquitin, Ubiquitination, Ubiquitin-like proteins (UBL) 1 Introduction The NF-κB transcription factors regulate expression of a large number of genes, which in turn drive a variety of physiological processes including innate and adaptive immunity, apoptosis, and cellular differentiation [1].

A) The dynamic of phosphorylation and degradation of IκB-α in total lysates. (b) The signal dependent ubiquitination of IκB-α associate with RelA. Maximal ubiquitination rate is observed between 10 and 15 min of stimulation and after dramatically decreased due to proteasomal degradation of IκB-α (30 min sample). RelA serves as loading controls. 1 Petro Starokadomskyy and Ezra Burstein Materials Reagents 1. Anti-RelA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2. Anti-phospho-IκB-α antibody Signaling, Danvers, MA, USA).

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