Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit #7325

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
SAPK/JNK Mouse mAb Coated Microwells
Phospho-SAPK/JNK (Thr183/Tyr185) Detection Antibody 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST'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 SAPK/JNK Mouse mAb has been coated onto the microwells. After incubating with cell lysate, total SAPK/JNK protein (non-phosphorylated and phosphorylated) is captured by the plate-bound antibody. Following extensive washing, Phospho-SAPK/JNK (Thr183/Tyr185) Antibody is added to detect the captured, phosphorylated fraction of SAPK/JNK protein. Anti-rabbit 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.

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

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, 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).

  1. Davis, R.J. (1999) Biochem Soc Symp 64, 1-12.
  2. Ichijo, H. (1999) Oncogene 18, 6087-93.
  3. Kyriakis, J.M. and Avruch, J. (2001) Physiol Rev 81, 807-69.
  4. Kyriakis, J.M. (1999) J Biol Chem 274, 5259-62.
  5. Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.
  6. Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem Sci 23, 481-5.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products