Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145

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

Kit Includes Volume Solution Color
P-c-Jun (S63) Rabbit Antibody Coated Microwells 96 tests
c-Jun Mouse Detection Antibody 11 ml Green
Anti-mouse 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 R

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

Description

CST's PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-c-Jun (Ser63) protein. A phospho-c-Jun (Ser63)-specific rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-c-Jun (Ser63) protein is captured by the coated antibody. Following extensive washing, c-Jun Mouse mAb is added to detect the captured phospho-c-Jun protein. HRP-linked anti-mouse antibody (#7076) 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-c-Jun (Ser63) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II detects endogenous levels of Phospho-c-Jun (Ser63) protein. As shown in Figure 1, using this Sandwich ELISA Kit #7145, a significant induction of phospho-c-Jun (Ser63) in UV-treated 293 cells can be detected. However, the level of total c-Jun protein (phospho and non-phospho), detected by the Total c-Jun Sandwich ELISA Kit II #7150, remains unchanged. Both C6 and NIH/3T3 cells treated with UV light or anisomycin show similar results (data not shown).This kit is more sensitive than kit #7260 (figure 2). 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 c-Jun at Ser63, detected by PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145, but does not affect the level of total c-Jun protein detected by PathScan® Total c-Jun Sandwich ELISA Kit II #7150. Lambda protein phosphatase (LPP) treatment of control cell lysates (37ºC for 90 minutes) abolished the basal phosphorylation of c-Jun in control lysates shown in both Sandwich ELISA and Western analysis. The OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-c-Jun (Ser63) Rabbit mAb #2378 (right panel) or c-Jun Rabbit mAb (6H5) #2374 (left panel), is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: Linear relationship between protein concentration of lysates from UV-treated 293 cells and kit assay optical density readings. Pathscan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145 is more sensitive than kit # 7260 over a range of lysate concentrations. 293 cells (70-90% confluence) were treated without or with UV and lysed after incubation at 37oC for 30 minutes.

Background

c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).

  1. Jochum, W. et al. (2001) Oncogene 20, 2401-12.
  2. Davis, R.J. (2000) Cell 103, 239-52.
  3. Hilberg, F. et al. (1993) Nature 365, 179-81.
  4. Raivich, G. et al. (2004) Neuron 43, 57-67.
  5. Behrens, A. et al. (2002) EMBO J 21, 1782-90.
  6. Riera-Sans, L. and Behrens, A. (2007) J Immunol 178, 5690-700.
  7. Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.
  8. Shaulian, E. and Karin, M. (2002) Nat Cell Biol 4, E131-6.
  9. Weiss, C. and Bohmann, D. (2004) Cell Cycle 3, 111-3.
  10. Karamouzis, M.V. et al. (2007) Mol Cancer Res 5, 109-20.
  11. Kim, S. and Iwao, H. (2003) J Pharmacol Sci 91, 177-81.
  12. Dass, C.R. and Choong, P.F. (2008) Pharmazie 63, 411-4.

Application References

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Protocols

Companion Products

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

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

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