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High‐throughput detection of nuclear factor‐kappaB activity using a sensitive oligo‐based chemiluminescent enzyme‐linked immunosorbent assay
Author(s) -
Bhattacharya Nupur,
Sarno Antonio,
Idler Irina S.,
Führer Marita,
Zenz Thorsten,
Döhner Hartmut,
Stilgenbauer Stephan,
Mertens Daniel
Publication year - 2010
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.25054
Subject(s) - relb , transcription factor , chemiluminescence , electrophoretic mobility shift assay , high throughput screening , oligonucleotide , microbiology and biotechnology , signal transduction , biology , luciferase , chemistry , dna , cell culture , p50 , biochemistry , genetics , transfection , chromatography , gene
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 (NFκB) 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 NFκB 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. Co‐ELISA is a sensitive, fast and cost‐efficient quantitation method for all DNA‐binding NFκB proteins that can be used in high‐throughput experimentation.