Description: The InstantOne™ ELISA is specifically engineered for accurate measurement of phosphorylated human, mouse, and rat NFkB p65 (Ser536), IkBa (Ser32/36), and IKKa (Ser176/180) in cell lysates. The InstantOne™ ELISA kit allows for fast analysis of samples in approximately one hour. All reagents used in a traditional sandwich ELISA are added in solution to a plate followed by a wash step and detection with the TMB colorimetric substrate.
IkappaB kinases, IKKa (IKK1) and IKKß (IKK2), phosphorylate and trigger degradation of the cytoplasmic NFkB inhibitors IkB (IkB alpha, IkB beta, and IkB gamma). These events result in the release of NFkB proteins from its inhibitory complex in the cytosol and enables NFkB to translocate into the nucleus to regulate transcription.
IkB proteins are present within the cytosol where they bind to the NFkB/Rel transcription factor complex to maintain its inactive state. To activate NFkB, IkB must dissociate from the complex. This is induced by phosphorylation of IkB at Ser32 and Ser36 in response to various extracellular signals, including inflammatory cytokines, growth factors, and chemokines. Phosphorylation targets IkB for ubiquitination and proteosomal degradation, thereby enabling NFkB to translocate into the nucleus to modulate gene expression. Examination of IkB phosphorylation has been used widely as an indicator of NFkB activation.
Nuclear Factor kappa B (NFkB) constitutes a family of transcription factors consisting of five members, including Rel (c-Rel), RelA (p65), RelB, NFkB1 (p50 and its precursor p105), and NFkB2 (p52 and its precursor p100). NFkB members can exist as either homo- or heterodimers. NFkB dimers containing p65 are activators of transcription. In unstimulated cells, NFkB is inactive and retained in the cytoplasm due to binding by inhibitory IkB proteins. Upon stimulation by inducers such as TNFa, IL-1, or PMA, IkBa is phosphorylated and degraded. This results in the release of the NFkB complex from the IKK complex and cleavage of the p105 subunit into its active p50 form. Subsequently, the p50/p65 complex translocates to the nucleus where it activates transcription. NFkB regulates the expression of numerous genes, including IkBa, cytokines, chemokines, adhesion targets, and acute phase proteins involved in growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters.