Product Overview :
NF-κB is a transcription factor that plays a pivotal role in a number of physiological cell processes such as growth, apoptosis, and immune response through regulating the expression of a number of cellular genes. Normally, NF-κB is inactive and retained in the cytoplasm bound to its inhibitory protein IκB. Upon stimulation of various factors such as infection of pathogens including viruses and bacteria or treatment of cytokines such as IL-1 or TNF-α, IκB proteins are phosphorylated, ubiquitinated and finally degraded. Released NF-κB proteins then form homo- or heterodimers, most commonly p50/p65 or p50/p50, which are transported into the nucleus where they bind to specific DNA sequences of targeted genes and activate transcription. NF-κB also induces expression of its own inhibitor, IκBα, which binds to active NF-κB in the nucleus to terminate the induction of gene expression. The NF-κB p65 Transcription Factor-Activity Assay kit is a non-radioactive transcription factor assay with an ELISA format. It offers an easy, speedy, sensitive and high-throughput method to detect the activation of transcription factors. In 96-well plates, double stranded oligonucleotides containing NF-κ B binding sequence have been coated. These oligonucleotides specifically capture the active NF-κB p65 contained in whole cell lysate or nuclear extracts after a short incubation. The specificity of the reaction between active NF-κB p65 and the DNA probe is additionally stringent because of the establishment of specific competitive DNA and non-specific competitive DNA probes in this reaction system.
Storage :
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2-8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
Kit Components :
NF-κB p65 DNA Probe Microplate, 96 wells (12 strips X 8 wells) coated with NF-κB probes, 1 plate
DNA Binding Buffer, 5X concentrated Buffer, 4 ml
Positive Control, Cell nuclear extracts, 1 vial (20 µl)
Specific Competitor DNA Probe, Free DNA probes that compete directly with the coated NF-κB probes. Can bind activated NF-κB, 1 vial
Non-specific Competitor DNA Probe, Free DNA probes with mutations of the coated DNA probe. Cannot bind activated NF-κB, 1 vial
Assay Reagent, 1X solution, 1 vial (200 µl)
DTT, 300 mM DTT, 1 vial (200 µl)
Wash Buffer Concentrate (20X), 20X concentrated solution, 25 ml
NF-κB p65 Primary Antibody, Anti-NF-κB p65 antibody, 1 vial
HRP-conjugated Secondary Antibody, Anti-IgG HRP conjugated antibody, 1 vial
Antibody Diluent Buffer, Buffer solution for diluting primary and secondary antibodies, 25 ml
TMB One-Step Substrate Reagent, 3,3,5,5'-tetramethylbenzidine (TMB) in buffer solution, 12 ml
Stop Solution, 0.2 M sulfuric acid, 8 ml
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