Figure 1: Treatment of 293 cells with UV stimulates phosphorylation of SAPK/JNK at Thr202/Tyr204, detected by PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA kit #7325, but does not affect the level of total SAPK/JNK protein detected by PathScan® Total SAPK/JNK Sandwich ELISA kit #7330. OD450 readings are shown in the top figure, while the corresponding western blot using Phospho-SAPK/JNK (Thr183/Tyr185) Antibody #9251 (right panel) or SAPK/JNK Rabbit mAb #9258 (left panel), is shown in the bottom figure.
Figure 2: Linear relationship between protein concentration of lysates from control and UV-treated 293 cells and kit assay optical density readings. 293 cells (80% confluence) were treated with UV and lysed after incubation at 37oC for 30 minutes.
|Product Includes||Volume||Solution Color|
|Phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb Coated Microwells||96 tests|
|SAPK/JNK Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® ST’s PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that
detects endogenous levels of Phospho-SAPK/JNK (Thr183/Tyr185) protein. A Phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb has been coated onto the microwells. After incubating with cell lysate, phospho-SAPK/JNK (Thr183/Tyr185) protein is captured by the plate-bound antibody. Following extensive washing, total SAPK/JNK (L7E7) Mouse mAb is added to detect the captured (non-phsophorylated and phosphorylated) total SAPK/JNK. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-SAPK/JNK protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit detects endogenous levels of Phospho-SAPK/JNK protein. Using this Sandwich ELISA Kit #7325, a significant induction of phospho-SAPK/JNK in 293 cells treated with UV is detected. However, the level of total SAPK/JNK (phospho and non-phospho), detected by PathScan® Total SAPK/JNK Sandwich ELISA Kit #7330, remains unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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 gamma radiation, ceramides, inflammatory cytokines, and in some instances, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs phosphorylate 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 in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).
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