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phospho-IκB (Ser32) STAR ELISA Kit
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PRODUCT FAMILY INFORMATION
STAR ELISA Kits
Upstate STAR (Signal Transduction Assay Reaction) ELISA kits are a fast, sensitive method to detect the relative amounts of total and activated targets through the use of phospho-specific antibodies.
Upstate STAR (Signal Transduction Assay Reaction) ELISA kits are a fast, sensitive method to detect the relative amounts of total and activated targets through the use of phospho-specific antibodies.
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Description:
phospho-IκB (Ser32) STAR ELISA Kit
Trade Name:
Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
I. TEST PRINCIPLE:
The colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The IκB plate is coated with a specific mouse monoclonal IκB capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing IκB antigen to be captured in the plate wells. The plate is then washed to remove any un-bound nonspecific material. A specific rabbit anti-phospho-IκB antibody is added to detect the captured IκB on the plate well that is phosphorylated on Ser32. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to generate a standard curve for IκB measurement.
II. BACKGROUND:
NFκB signaling is a critical regulator not only of immune function, but also of proliferation versus apoptosis in response to various stimuli. In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins that prevent it from entering the nucleus, thus keeping NFκB in an inactive state. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFα, IL-1, or PMA, IκBα is rapidly phosphorylated by IKK (IκB Kinase), which phosphorylates IκB on serines 32 and 36, allowing recognition by the ubiquitin ligase machinery, leading to its polyubiquination and degradation, resulting in the release of the NFκB complex. Once IκB is degraded, NFκB is able to initiate transcription. The NFκB complex then translocates to the nucleus where it activates gene transcription. NFκB induces the transcription of its own inhibitor, IκBα, causing an autoregulatory mechanism of NFκB activity and generating the inactive form of NFκB. The newly formed nuclear NFκB- IκBα complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFκB complexes primed for another round of activation to take place. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes. Mutant IκB in which serines 32 and 36 are changed to alanines, is not phosphorylated, and therefore not degraded. Cells expressing this protein are not able to activate NFκB, providing a useful tool to study the role of NFκB in various pathways and processes.
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