PathScan® MAP Kinase Multi-Target Sandwich ELISA Kit #7274
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Supporting Data
| REACTIVITY | H M |
Application Key:
- ELISA-ELISA
Species Cross-Reactivity Key:
- H-Human
- M-Mouse
Product Information
Product Description
CST's PathScan® MAP Kinase 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 phospho-p44/42 MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), MEK1, phospho-MEK1 (Ser217/221), SAPK/JNK and phospho-SAPK/JNK (Thr183/Tyr185). These molecules represent convergence points and key regulatory proteins in signaling pathways controlling cellular events such as growth, differentiation and the response to stress and inflammation. 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 target protein is captured by the coated antibody. 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.
Protocol
Specificity / Sensitivity
CST's PathScan® MAP Kinase Multi-Target Sandwich ELISA Kit #7274 detects endogenous levels of six proteins: phospho-p44/42 MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), MEK1, phospho-MEK1 (Ser217/221), SAPK/JNK and phospho-SAPK/JNK (Thr183/Tyr185). Differential phosphorylation of these proteins can be observed over time in response to various growth factor and 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.
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
Background
Both p44 and p42 MAP kinases (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation (1-6). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (Thr202 and Tyrr204 of human MAP kinase or Thr183 and Tyr185 of rat MAP kinase) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (7,8).
MEK1 and MEK2 are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation. Activation of MEK1 and MEK2 occurs through phosphorylation of Ser217 and Ser221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (1,9,10).
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 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 (15-20). 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 (16). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (17). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (17). 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 (18).
MEK1 and MEK2 are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation. Activation of MEK1 and MEK2 occurs through phosphorylation of Ser217 and Ser221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (1,9,10).
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 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 (15-20). 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 (16). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (17). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (17). 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 (18).
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