The stress-activated protein kinase/Jun- amino-terminal kinase SAPK/JNK is potently and prefer- entially activated by a variety of environmental stresses, including UV and gamma radiation, ceramides, inflamma- tory 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). SAPK/JNK, when active as a dimer, can translocate to the nucleus where it regulates transcription through its ef- fects 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 Phospho-SAPK/JNK (Thr183/Tyr185) Detection Antibody and Anti-rabbit IgG, HRP-conjugated Antibody. HRP substrate, TMB, is added for color develop- ment. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-SAPK/JNK (Thr183/Tyr185) protein.
B. Reagents Required But Not Supplied:1. Coating Buffer: 1X PBS, (20X PBS) 3.2 mM Na 2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4 2. Wash Buffer: 1X PBS/0.05% Tween-20, (20X PBST) 3. Blocking Buffer: 1X PBS/0.05% Tween-20, 1% BSA 4. 1X Cell Lysis Buffer: (10X Cell Lysis Buffer) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylene diamine tetraacetate (EDTA), 1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin. 5. TMB Substrate: (TMB Substrate) 6. STOP Solution: (STOP Solution) NOTE: Reagents should be made fresh daily
C. Storage and StabilityStore capture and detection antibodies at 4°C. Store anti-mouse IgG (HRP) at -20°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
