Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276

Kit Includes Volume Solution Color Cap Color
NF-κB p65 Rabbit Antibody Coated Microwells 16 tests
NF-κB p65 Mouse Detection Antibody 1.8 mlGreenGray
Anti-Rabbit IgG HRP-Linked Antibody 1.8 mlRedGray
NF-κB p65 Mouse Antibody Coated Microwells 16 tests
Phospho-NF-κB p65 (S536) Rabbit Detection Antibody 1.8 mlGreenOrange
Anti-rabbit IgG HRP-Linked Antibody 1.8 mlRedOrange
SAPK/JNK Mouse Antibody Coated Microwells 16 tests
Phospho-SAPK/JNK (Thr183/Tyr185) Rabbit Detection Antibody 1.8 mlGreenPurple
Anti-rabbit IgG HRP-Linked Antibody 1.8 mlRedPurple
Phospho-p38 MAP Kinase (T180/Y182) Mouse Antibody Coated Microwells16 tests
p38 MAPK Rabbit Detection Antibody1.8 mlGreenYellow
Anti-Rabbit IgG HRP-Linked Antibody1.8 mlRedYellow
Stat3 Rabbit Antibody Coated Microwells 16 tests
Phospho-Stat3 (Tyr705) Mouse Detection Antibody 1.8 mlGreenDark Pink
Anti-Mouse IgG HRP-Linked Ab 1.8 mlRedDark Pink
IκB-α Mouse Antibody Coated Microwells 16 tests
Phospho-IκB-α (Ser32) Rabbit Detection Ab 1.8 mlGreenBlue
Anti-rabbit IgG HRP-Linked Antibody 1.8 mlRedBlue
TMB Substrate 11 mlColorless
STOP Solution 11 mlColorless
Sealing Tape 2 sheets
20X Wash Buffer 25 mlColorless
Sample Diluent 25 mlBlue
Cell Lysis Buffer (10X) #980315 mlYellowish

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 M

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

Description

CST's PathScan® Inflammation Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of NF-κB p65, phospho-NF-κB p65 (Ser536), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-IκB-α (Ser32). These molecules represent convergence points and key regulatory proteins in signaling pathways controlling the stress and inflammation response. Sixteen tests are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits**. Briefly, a capture antibody* has been coated onto the microwells. After incubation with cell lysates, the coated antibody captures the target protein. Following extensive washing, a detection antibody* is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein. *Antibodies in kit are custom formulations specific to kit. **See companion products.

Specificity / Sensitivity

CST's PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276 detects endogenous levels of six proteins: NF-κB p65, phospho-NF-κB p65 (Ser536), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-IκB-α (Ser32). Differential activation of these proteins can be observed over time in response to various cytokine treatments, as shown in Figure 1. The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA kits**. **See companion products. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Sandwich ELISA

Sandwich ELISA

Figure 1. Treatment of HeLa cells with TNFα/IL-1β (A) or IL-6 (B) induces differential phosphorylation of NF-κB p65 at Ser536, SAPK/JNK at Thr183/Tyr185, p38 MAPK at Thr180/Tyr182, Stat3 at Tyr705 and IκB-α at Ser32 as detected by the PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276. While dynamic phosphorylation is observed throughout the time course, the level of total NF-κB p65, SAPK/JNK, p38 MAPK, Stat3 and IκB-α remains unchanged as demonstrated by sandwich ELISA and Western analysis. HeLa cells (80-90% confluent) were serum starved and stimulated with IL-6 (100 ng/mL) for 5, 10, 20, 40 and 80 minutes at 37ºC. Alternatively, exponentially growing cultures of HeLa (80-90% confluent) were treated simultaneously for the indicated times at 37ºC with 20 ng/mL TNF-α and 10 ng/mL IL-1β. Lysates were assayed at a protein concentration of 0.5 mg/mL. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left figure, while the corresponding Western blots are shown in the right figure. The antibodies used for the Western analyses include NF-κB p65 Antibody #3034, Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033, SAPK/JNK (56G8) Rabbit mAb #9258, Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb #9255, p38α MAP Kinase Antibody #9218, Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb #9216, Stat3 Antibody #9132, Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138, IκB-α Antibody #9242 and Phospho-IκBα (Ser32) (14D4) Rabbit mAb #2859.

Sequence

Sequence

Figure 2. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of NF-κB p65 (gray; 1 & 2), Phospho-NF-κB p65 (Ser536) (orange; 3 & 4), Phospho-SAPK/JNK (Thr183/Tyr185) (purple; 5 & 6), Phospho-p38 MAPK (Thr180/Tyr182) (yellow; 7 & 8), Phospho-Stat3 (Tyr705) (dark pink; 9 & 10) and Phospho-IκB-α (Ser32) (blue; 11 & 12).

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammation, stress and immune responses. There are five family members in mammals: RelA/p65, c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). These proteins function as dimeric transcription factors. In unstimulated cells, NF-κB/Rel proteins are sequestered in the cytoplasm and inhibited by the IκB proteins. NF-κB-activating agents induce phosphorylation of IκB's, targeting them for degradation and thereby releasing the NF-κB/Rel complexes. Active NF-κB/Rel complexes are further activated by phosphorylation (1-4).The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is 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 (5-10). As with the other MAPKs, the core-signaling unit is composed of a MAPKKK, typically MEKK1-4, or by a mixed lineage kinase (MLK), which phosphorylates and activates MKK4-7, which then phosphorylates Thr183 and Tyr185 to activate the SAPK/JNK kinase (6). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (7). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (7). 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 (8).p38 MAP kinase (MAPK) participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokines and a variety of cellular stresses. MKK3, MKK6 and SEK (MKK4) activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182 (11-14).The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors. Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation and DNA binding (15,16).

  1. Ghosh, S. and Karin, M. (2002) Cell 109 Suppl, S81-96.
  2. DiDonato, J. et al. (1996) Mol Cell Biol 16, 1295-304.
  3. Sakurai, H. et al. (1999) J Biol Chem 274, 30353-6.
  4. Mattioli, I. et al. (2004) J Immunol 172, 6336-44.
  5. Davis, R.J. (1999) Biochem Soc Symp 64, 1-12.
  6. Ichijo, H. (1999) Oncogene 18, 6087-93.
  7. Kyriakis, J.M. and Avruch, J. (2001) Physiol Rev 81, 807-69.
  8. Kyriakis, J.M. (1999) J Biol Chem 274, 5259-62.
  9. Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.
  10. Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem Sci 23, 481-5.
  11. Raingeaud, J. et al. (1995) J Biol Chem 270, 7420-6.
  12. Raman, M. et al. (2007) Oncogene 26, 3100-12.
  13. Zarubin, T. and Han, J. (2005) Cell Res 15, 11-8.
  14. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
  15. O'Shea, J.J. et al. (2002) Cell 109 Suppl, S121-31.
  16. Kaptein, A. et al. (1996) J Biol Chem 271, 5961-4.

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

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

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

Products