High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay

International Journal of Cancer. Journal International Du Cancer
Nupur BhattacharyaDaniel Mertens

Abstract

Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, nuclear factor-kappaB (NFkappaB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide-based enzyme-linked immunosorbent assay (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkappaB components (i.e., RelA, p50, p52, RelB and c-Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co-ELISA has a 3.5- to 43-fold higher signal-over-noise ratio than currently available assays, whereas the percent relative standard deviation is 3- to 6-fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. ...Continue Reading

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Citations

Jan 17, 2016·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Zaid H MaayahAyman O S El-Kadi
Oct 31, 2015·Genes, Brain, and Behavior·Cathy L Barr, Virginia L Misener
Dec 23, 2011·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Karen F UnderwoodAntonino Passaniti
Feb 11, 2018·Scientific Reports·Zaid H MaayahAyman O S El-Kadi
Jun 3, 2015·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Hassan N AlthurwiAyman O S El-Kadi
Jul 24, 2013·The Journal of Immunology : Official Journal of the American Association of Immunologists·Takuya MatsumotoMasaki Yasukawa

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