p38 MAPK Activation Kit

• A more biologically relevant measure of kinase activation relative to Western blots or in vitro kinase assays
• No cell lysis, purification, or filtration steps
• Immunofluorescence based assay provides sensitive results without radioactivity
• Kit antibody preferentially binds to dually phosphorylated (fully active) p38, with minimized cross-reactivity to other MAPKs
• Manual sample preparation/processing requires approximately 3 hours post-compound treatment

The p38 MAPK Activation Kit provides the reagents and protocol necessary to quantify p38 MAPK activation by directly measuring its translocation from the cytoplasm to the nucleus. The assay is performed on live cells growing on standard high-density microplates. The core reagents, a primary p38 MAPK antibody and DyLight™ 488-conjugated Secondary Antibody, are supplied. The nuclear region is identified by the nuclear dye, Hoechst, also included in the kit.

The MAPK super family comprises c-Jun N-terminal kinases (JNKs), also known as stress activated protein kinases (SAPKs), p38 kinases, and extra cellular signal regulated kinases (ERKs). The p38 signal transduction pathway has been associated with the regulation of vital cellular processes including cell growth, division, differentiation, inflammation, and death. p38 is activated by bacterial lipopolysaccharide, physiochemical changes in the extra cellular milieu (heat, hyper- and hypo- osmolarity, UV irradiation, sodium arsenite, and anisomycin, and proinflammatory cytokines (e.g., IL-1 and tumor necrosis factor-alpha).  p38 MAPK is in a pathway comprising a cascade of kinases, whereby p38 acts as a substrate of one or more MKK kinases. Once activated by phosphorylation, p38 phosphorylates one or more substrates, leading to transcriptional changes and other diverse cellular processes. For example, the differentiation of neuronal-like precursor cells such as PC12 cells and the myogenesis of C2C12 cells involve the activation (phosphorylation) of p38. Viral infection also induces p38 activity.

The optimized protocol is for a fixed end-point immnunofluorescent assay. Inhibitors of p38 MAPK translocation are screened by stimulating cells with a control inducer, such as TNFα, after exposing live cells to test compounds. To identify agonists of p38 MAPK translocation, TNF α is replaced with test compounds. Translocation is directly quantified as the difference in cytoplasmic to nuclear intensity of the labeled kinase. The p38 MAPK Activation Kit, in combination with the ArrayScan HCS System and the Cytoplasm to Nucleus Translocation Application software enables automated plate handling, focusing, cell image acquisition, analysis and quantification of p38 MAPK activation. For a more detailed description of the image-processing algorithm, see the Cytoplasm to Nucleus Translocation Application Guide that accompanies the Cytoplasm to Nucleus Translocation Application software.

Figure 1. Images of fluorescently labeled HeLa cells before (left) and after (right) activation by p38 MAPK by TNF α (100 ng/mL for 15 min). Cells were prepared according to the kit protocol. Nuclei appears blue (Hoechst) and ERK appears green (Alexa Fluor® 488). Images were aquired using the ArrayScan® HCS reader.

Figure 2. Principle of the p38 MAPK Activation Kit product. p38 MAPK (green) normally present in the cytoplasm, translocates to the nucleus upon activation by an inducer such as TNF α.

Figure 3. Dose dependence of TNF-α induced p38 activation in HeLA cells. Error bars represent Mean ± SD for 8 wells of a 96-well microplate.

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