News from the Bench

Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.

Subscribe

Important Ordering Details

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

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

Questions?

Find answers on our FAQs page.

ANSWERS  

Visit PhosphoSitePlus®

PTM information and tools available.

LEARN MORE

REACTIVITY
Product Includes Volume Solution Color
NF-kB p65 Mouse Antibody Coated Microwells 16 tests
NF-kB p65 Rabbit Detection Antibody 1.8 ml Green
Anti-rabbit IgG, HRP-linked Antibody 1.8 ml Red
Phospho-NF-κB p65 (S536) Mouse Antibody Coated Microwells 16 tests
NF-κB p65 Rabbit Detection Antibody 1.8 ml Green
Anti-rabbit IgG, HRP-linked Antibody 1.8 ml Red
Phospho- SAPK/JNK (Thr183/Tyr185) Rabbit Antibody Coated Microwells 16 tests
SAPK/JNK (L7E7) Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
Phospho-p38 MAP Kinase (T180/Y182) Mouse Antibody Coated Microwells 16 tests
p38 MAPK Rabbit Detection Antibody 1.8 ml Green
Anti-rabbit IgG, HRP-linked Antibody 1.8 ml Red
Stat3 Rabbit Antibody Coated Microwells 16 tests
Phospho-Stat3 (Y705) Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
IκB-α Mouse Antibody Coated Microwells 16 tests
Phospho-IκB-α (S32) Rabbit Detection Antibody 1.8 ml Green
Anti-rabbit IgG, HRP-linked Antibody 1.8 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
Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) 9803 15 ml Yellowish

Product 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.


Species Reactivity: Human, Mouse

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.  Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.

2.  Ghosh, S. and Karin, M. (2002) Cell 109, S81-S96.

3.  DiDonato, J. et al. (1996) Mol Cell Biol 16, 1295-304.

4.  Ichijo, H. (1999) Oncogene 18, 6087-93.

5.  Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.

6.  Sakurai, H. et al. (1999) J Biol Chem 274, 30353-6.

7.  Kyriakis, J.M. and Avruch, J. (2001) Physiol Rev 81, 807-69.

8.  Mattioli, I. et al. (2004) J Immunol 172, 6336-44.

9.  Kyriakis, J.M. (1999) J Biol Chem 274, 5259-62.

10.  Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.

11.  Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.

12.  Davis, R.J. (1999) Biochem Soc Symp 64, 1-12.

13.  Raman, M. et al. (2007) Oncogene 26, 3100-12.

14.  Zarubin, T. and Han, J. (2005) Cell Res 15, 11-8.

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.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PathScan® is a trademark of Cell Signaling Technology, Inc.