If NF-kB activation could not be detected, is it possible that the cytokines will be produced?
I'm working with macrophage cells. Nitric oxide production can be detected after treating the cell with 1ug/mL of LPS. However, NF-kB activation cannot be detected using an ELISA kit. So, is it possible that cytokines such as IL-1, IL-6, TNF-a, IFN-g will be detected from the supernatant if NF-kB couldn't be detected from cell lysate?
I am thinking Why the effect was not found, Since LPS is a strong ligand for toll receptor-4 and we know tlr4 structural confirmation or disruption results cytoplasmic Ikk and MAPK subsequently know mechanism of translocan and gene expression.
With your Question ,i can say it is possible to assay from IKK(alpha, beta, gama) complex to Ubiquitination (only Ikb-alpha) product and nuclear import product (NF-kb p65 or50/52).
For NF-Kb imported complex to nucleus must be phosphorylated by kinase effect.
I can suggest some papers, that might be your solution: I red most of their papers, they are unknown masters of mine, perhaps scientific God fathers,
Sankar Ghosh, Shizuao akira, Laurent Boyer and http://www.bu.edu/nf-kb/physiological-mediators/inducers/
Bonizzi G. Karin M. (2004) The two NFkB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25, 280-288.
Hi Pui-Mun Chan
Check your ELISA kit detection limit which is important factor, Use western blotting of path hunting technique for detecting the NF-Kb more have more detection levels than ELISA so change your mode of detection you will definitely get the results. Also you can detect your cytokines IL-1, IL-6, TNF-a, IFN-gamma which is not related to NF-Kb detected by your Kit (mode of detection).
All the best
Thank you for all the response.
I was thinking to use RT-PCR to determine the gene expression of NF-KB since the lab I'm working in, has all the equipments for RT-PCR.
About the cytokines, I was actually a bit doubtful whether to do the cytokine assay since NF-kB activation is not detected and the cytokine ELISA kits is very expensive in Malaysia as I'm planning to use multiplex technology.
Check for Il-6/Tnfa gene expression...that will serve as a positive control for LPS treatment....good luck..
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