The stress-activated protein kinase/Jun- amino-terminal kinase (SAPK/JNK) is potently and preferentially activated by a variety of environmental stresses, including UV and g radiation, ceramides, inflammatory cytokines and in some instances, by growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1- 4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4-7, which then phosphory- late and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4-7 can be activated by a pathway independent of small GTPases via stimulation of a member of the germinal center kinase (GCK) family (4). There are three SAPK/JNK genes with further diversification resulting from alternative splicing (3). Active SAPK/JNK dimers can translocate to the nucleus to regulate transcription through its effects on c-Jun, ATF-2 and other transcription factors (3,5).
Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The SAPK/JNK Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by SAPK/JNK Detection Antibody and Anti-Rabbit IgG, HRP-conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of total SAPK/JNK protein.