ERK 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 quantitative and qualitative results without radioactivity
• Kit antibody preferentially binds to dually phosphorylated (fully active) ERK, with minimized cross-reactivity to other MAPKs
• Manual sample preparation/processing requires approximately 3 hours post-compound treatment

The ERK MAPK Activation Kit provides the reagents and protocol necessary to quantify ERK activation by directly measuring its translocation from the cytoplasm to the nucleus. This fixed end-point assay is performed on live cells growing on standard high-density microplates. Inhibitors of ERK translocation are screened by stimulating cells with a control inducer, such as phorbol 12-myristate 13-acetate (PMA) after exposing cells to the test compounds. The kit is supplied with an anti-ERK primary antibody and a DyLight™ 488-conjugated secondary antibody. The nuclear region is identified by the nuclear dye, Hoechst, also included in the kit.

Mitogen-activated protein kinase (MAPK) pathways regulate an extensive range of cellular processes including gene transcription, cytoskeletal organization, metabolic homeostasis, cell growth and apoptosis. MAPKs are activated by concomitant Tyr and Thr phosphorylation via a complex multi-step signal transduction cascade. The extracellular signal-regulated kinases (ERK) 1 and 2, also known as p44 and p42 MAPK, accumulate in the nucleus after stimulation with mitogens. Thus the MAPK/ERK cascade constitutes a functional signaling unit that links surface receptor-mediated signals to nuclear events.

The ERK MAPK Activation Kit enables ERK quantitation by its translocation to the nucleus. This kit in combination with the ArrayScan HCS Reader and the Cytoplasm to Nucleus Translocation BioApplication software allows automated plate handling, focusing, cell image acquisition and analysis of ERK MAPK activation. For a more detailed description of the image processing algorithm, see the Cytoplasm to Nucleus Translocation BioApplication Guide that accompanies the Cytoplasm to Nucleus Translocation BioApplication software.

Figure 1. Images of fluorescently lable NIH 3T3 cells before (left) and after (right) activation of ERK by PMA (100 ng/mL for 30 min.) Cells were prepared according to the kit protocol.  Nuclei appear blue (Hoechst) and ERK appears green (Alexa Fluor 488). Images were aquired by the ArrayScan HCS Reader.

Figure 3. Dose response curve for PMA in NIH 3T3 cells.  NIH 3T3 cells were stimulated with PMA for 30 minutes, then labeled as per the ERK MAPK Activation kit protocol.  The dose response curve was used to determine an EC₅₀ of 2.7 ng/mL.

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