Product Pathways - PathScan ELISA

PathScan® Total SAPK/JNK Sandwich ELISA Kit #7330

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Kit Includes	Volume	Solution Color
SAPK/JNK (7325-7E7) Mouse mAb Coated Microwells
SAPK/JNK Detection Ab	11 milliliters	Green
Anti-rabbit IgG HRP-Linked A	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
Sealing Tape	2 sheets
20X Wash Buffer	25 milliliters	Colorless
Sample Diluent	25 milliliters	Blue
Cell Lysis Buffer (10X) # 9803	15 milliliters	Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human

Description

CST's PathScan® Total SAPK/JNK Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total SAPK/JNK protein. A SAPK/JNK (7325-7E7) Mouse mAb* has been coated onto the microwells. After incubation with cell lysates, Both nonphospho- and phospho-SAPK/JNK proteins are captured by the coated antibody. Following extensive washing, a SAPK/JNK (56G8) Rabbit mAb #9258* is added to detect the captured SAPK/JNK protein. HRP-linked anti-rabbit antibody #7074* 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 total SAPK/JNK protein.* Antibodies in this kit are custom formulations specific to the kit.

Specificity / Sensitivity

CST's PathScan® Total SAPK/JNK Sandwich ELISA Kit detects endogenous levels of total SAPK/JNK protein. Using PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit #7325, a significant induction of phospho-SAPK/JNK in 293 cells treated with UV can be detected. However, the level of total SAPK/JNK (phospho and non-phospho), detected by this Sandwich ELISA Kit #7330, remains unchanged (Figure 1).

Background

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, 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 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 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).

1. Davis, R.J. (1999) Biochem. Soc. Symp. 64, 1-12.
2. Ichijo, H. (1999) Oncogene 18, 6087-6093.
3. Kyriakis, J.M. and Avruch, J. (2001) Physiol. Rev. 81, 807-869.
4. Kyriakis, J.M. (1999) J. Biol. Chem. 274, 5259-5262.
5. Leppa, S. and Bohmann, D. (1999) Oncogene 18, 6158-6162.
6. Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.

Application References

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Companion Products

• 7325 PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit
• 9251 Phospho-SAPK/JNK (Thr183/Tyr185) Antibody
• 9258 SAPK/JNK (56G8) Rabbit mAb
• 7074 Anti-rabbit IgG, HRP-linked Antibody

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.