Product Pathways - PathScan ELISA
PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272
|Kit Includes||Volume||Solution Color||Cap Color|
|Akt Rabbit Ab Coated Microwells||16 tests|
|Akt1 Mouse Detection Ab||1.8 ml||Green||Red|
|Anti-Mouse IgG HRP-Linked Ab||1.8 ml||Red||Red|
|Phospho-Akt (Ser473) Rabbit Ab Coated Microwells||16 tests|
|Akt1 Mouse Detection Ab||1.8 ml||Green||Tan|
|Anti-Mouse IgG HRP-Linked Ab||1.8 ml||Red||Tan|
|MEK1 Mouse Ab Coated Microwells||16 tests|
|Phospho-MEK1/2 (Ser217/221) Rabbit Detection Ab||1.8 ml||Green||Green|
|Anti-Rabbit IgG HRP-Linked Ab||1.8 ml||Red||Green|
|p38α MAP Kinase Rabbit Ab Coated Microwells||16 tests|
|Phospho-p38α MAPK (Thr180/Tyr182) Mouse Detection Ab||1.8 ml||Green||Yellow|
|Anti-Mouse IgG HRP-Linked Ab||1.8 ml||Red||Yellow|
|Stat3 Rabbit Ab Coated Microwells||16 tests|
|Phospho-Stat3 (Tyr705) Mouse Detection Ab||1.8 ml||Green||Dark Pink|
|Anti-Mouse IgG HRP-Linked Ab||1.8 ml||Red||Dark Pink|
|NF-κB p65 Mouse Ab Coated Microwells||16 tests|
|Phospho-NF-κB p65 (Ser536) Rabbit Detection Ab||1.8 ml||Green||Orange|
|Anti-Rabbit IgG HRP-Linked Ab||1.8 ml||Red||Orange|
|TMB Substrate||11 ml||Colorless|
|STOP Solution||11 ml||Colorless|
|Sealing Tape||2 sheets|
|20X Wash Buffer||25 ml||Colorless|
|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 (10X), which is stored at –20°C (packaged separately).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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-p38 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-p38 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.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.
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 37ºC. 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 37ºC 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, p38 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.
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-p38 MAPK (Thr180/Tyr182) (yellow; 7 & 8), Phospho-Stat3 (Tyr705) (dark pink; 9 & 10) and Phospho-NF-κB p65 (Ser536) (orange; 11 & 12).
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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- 7170 PathScan® Total Akt1 Sandwich ELISA Kit
- 7160 PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit
- 7175 PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA Kit
- 7300 PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit
- 7173 PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kit
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 7946 PathScan® Phospho-p38 MAPK (Thr180/Tyr182) Sandwich ELISA Kit
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.