By Michael J. May

Focusing on crucial perception into the tools and methods required to dissect the advanced mechanisms of NF-κB activation, law, and serve as. NF-kappa B: equipment and Protocols guides readers via commonplace techniques to realize NF-κB pathway activation, detection and research of NF-κB signaling, and strategies to review the keep watch over of NF-κB signaling. As a quantity within the hugely profitable Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible protocols, and pointers on troubleshooting and heading off identified pitfalls.

Comprehensive and authoritative, NF-Kappa B: equipment and Protocols provides a well timed and important source for researchers trying to practice experiments aimed toward realizing the function of NF-κB signaling in future health and disease.

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1–5 µg of pGL3-Basic luciferase reporter vector must be digested with the same restriction enzymes. Repeat steps 1–3 above using the pGL3-Basic vector. 5. Quantify the promoter fragment and digested pGL3-Basic using a NanoDrop spectrometer, and, with the following formula, calculate the required quantities of insert (promoter) fragment and linearized vector (digested pGL3-Basic) required for the ligation reaction (see Note 19). Size( kb ) insert Size( kb ) vector ´ ng of vector = ng of insert required for a1 : 1molar ratio 6.

In this regard, IκB phosphorylation and degradation can be detected using a number of commercially available antibodies. However, monitoring IκB ubiquitination in vivo is more challenging due to rapid degradation of the target. Analysis of IκB Degradation and Ubiquitination 17 In this article, we review the methods to monitor IκB degradation and a protocol to monitor IκB-α ubiquitination in cells, first developed by Ben-Neriah and colleagues [4] and subsequently used in our laboratory, to study degradation and ubiquitination kinetics within the context of disease-causing mutations affecting the NF-κB pathway.

5. Thermal cycler. 6. Luminometer. 2 Cell Culture 1. 4 murine macrophage cell line. 2. Cell culture medium of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin, 2 mM L-glutamine, and 10 % (volume/volume) fetal bovine serum (FBS). Store at 4 °C until required. Pre-warm medium to 37 °C prior to culturing of cells. 3. 24-well tissue culture dishes. 4. Cell scraper. 3 Promoter Cloning 1. Sterile DNase-free pipette tips. 2. 5-ml microfuge tubes.

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