Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272

Kit IncludesVolumeSolution ColorCap Color
Akt Rabbit Ab Coated Microwells 16 tests
Akt1 Mouse Detection Ab 1.8 millilitersGreenRed
Anti-Mouse IgG HRP-Linked Ab 1.8 millilitersRedRed
Phospho-Akt (Ser473) Rabbit Ab Coated Microwells 16 tests
Akt1 Mouse Detection Ab 1.8 millilitersGreenTan
Anti-Mouse IgG HRP-Linked Ab 1.8 millilitersRedTan
MEK1 Mouse Ab Coated Microwells 16 tests
Phospho-MEK1/2 (Ser217/221) Rabbit Detection Ab 1.8 millilitersGreenGreen
Anti-Rabbit IgG HRP-Linked Ab 1.8 millilitersRedGreen
p38α MAP Kinase Rabbit Ab Coated Microwells 16 tests
Phospho-p38α MAPK (Thr180/Tyr182) Mouse Detection Ab 1.8 millilitersGreenYellow
Anti-Mouse IgG HRP-Linked Ab 1.8 millilitersRedYellow
Stat3 Rabbit Ab Coated Microwells 16 tests
Phospho-Stat3 (Tyr705) Mouse Detection Ab 1.8 millilitersGreenDark Pink
Anti-Mouse IgG HRP-Linked Ab 1.8 millilitersRedDark Pink
NF-κB p65 Mouse Ab Coated Microwells 16 tests
Phospho-NF-κB p65 (Ser536) Rabbit Detection Ab 1.8 millilitersGreenOrange
Anti-Rabbit IgG HRP-Linked Ab 1.8 millilitersRedOrange
TMB Substrate 11 millilitersColorless
Stop Solution 11 millilitersColorless
Sealing Tape 2 sheets
20X Wash Buffer 25 millilitersColorless
Sample Diluent 25 millilitersBlue
Cell Lysis Buffer (10X) #980315 millilitersYellowish

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

Description

CST's PathScan® Signaling Nodes 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 Akt1, phospho-Akt1 (Ser473), phospho-MEK1 (Ser217/221), phospho-p38α MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-NF-κB p65 (Ser536). These molecules represent convergence points and key regulatory proteins in signaling pathways controlling cellular events such as growth and differentiation, energy homeostasis, 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.

Specificity / Sensitivity

CST's PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272 detects endogenous levels of six proteins: Akt1, phospho-Akt1 (Ser473), phospho-MEK1 (Ser217/221), phospho-p38α MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-NF-κB p65 (Ser536). Differential phosphorylation of these proteins can be observed over time in response to various growth factor and cytokine treatments. As shown in Figure 1, stimulation of serum-starved NIH/3T3 cells with PDGF generates robust phosphorylation of Akt1 at Ser473 and MEK1 at Ser217/221 but relatively weak phosphorylation of Stat3 at Tyr705. Simultaneous treatment of NIH/3T3 cells with TNF-α and IL-1β results in phosphorylation of Akt1 at Ser473, MEK1 at Ser217/221, p38α MAPK at Thr180/Tyr182 and NF-κB p65 at Ser536 but no activation of Stat3. By comparison, NIH/3T3 cells respond to IL-6 primarily through the activation of Stat3 rather than Akt1, MEK1, p38α MAPK or NF-κB p65. The level of each target protein (phospho and nonphospho) remains unchanged throughout the 80 minute time course as demonstrated by Western analysis.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.

Sandwich ELISA

Sandwich ELISA

Figure 1. Treatment of NIH/3T3 cells with PDGF (A), TNFα/IL-1β (B) or IL-6 (C) induces differential phosphorylation of Akt1 at Ser473, Stat3 at Tyr705, p38α MAPK at Thr180/Tyr182, MEK1 at Ser217/221 and NF-κB p65 at Ser536 as detected by the PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272. While dynamic changes in phosphorylation are observed throughout the time course, the level of total Akt, MEK1, Stat3, p38α MAPK and NF-κB p65 remains unchanged as demonstrated by sandwich ELISA and Western analysis. NIH/3T3 cells (80-90% confluent) were starved overnight and stimulated with either PDGF (100 ng/mL) or IL-6 (100 ng/mL) for 5, 10, 20, 40 and 80 minutes at 37°C. For simultaneous treatment with TNF-α and IL-1β, exponentially growing cultures of NIH/3T3 (80-90% confluent) were treated for the indicated times at 37°C with 20 ng/mL TNF-α and 10 ng/mL IL-1β. Lysates were assayed at a protein concentration of 0.45 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 Akt Antibody #9272, Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058, MEK1 (61B12) Mouse mAb #2352, Phospho-MEK1/2 (Ser217/221) Antibody #9121, Stat3 Antibody #9132, Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138, p38α MAP Kinase Antibody #9218, Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb #9216, NF-κB p65 Antibody #3034 and Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033.

Table

Table

Figure 2. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of Akt1 (red; 1 & 2), Phospho-Akt1 (Ser473) (tan; 3 & 4), Phospho-MEK1 (Ser217/221) (green; 5 & 6), Phospho-p38α MAPK (Thr180/Tyr182) (yellow; 7 & 8), Phospho-Stat3 (Tyr705) (dark pink; 9 & 10) and Phospho-NF-κB p65 (Ser536) (orange; 11 & 12).

Background

Akt is a protooncogene with a critical regulatory role in diverse cellular processes including growth, survival and the cell cycle. Akt is also a major regulator of insulin signaling and glucose metabolism (1-4). Akt is activated by PI3 kinase signaling and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation withing the carboxyl terminus at Ser473 by PDK2 (5-7).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 serine 217 and serine 221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (8-10).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 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).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 (17-20).

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