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High-throughput detection of NFkappaB activity using a sensitive oligo-based ... Related Articles

High-throughput detection of NFkappaB activity using a sensitive oligo-based chemiluminescent ELISA.

Int J Cancer. 2009 Nov 18;

Authors: Bhattacharya N, Sarno A, Idler IS, Führer M, Zenz T, Döhner H, Stilgenbauer S, Mertens D

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, 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 ELISA (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, while the percent relative standard deviation is 3-to 6-fold lower. Also, the novel method is faster than EMSA, not restricted to transfectable cells as is the case for luciferase reporter assays, and ten times more cost-efficient than commercially available ELISA assays. Co-ELISA is a sensitive, fast, and cost-efficient quantitation method for all DNA-binding NFkappaB proteins that can be used in high-throughput experimentation. (c) 2009 UICC.

PMID: 19924814 [PubMed - as supplied by publisher]