Product Pathways - Screening Technologies
PhosphoPlus® p44/42 MAPK (Erk1/2)(Thr202/Tyr204) In-Cell Duet (ICW Compatible) #7263
|7263L||1 Kit (5 X 96 well plates)||---||In Stock||---|
|7263S||1 Kit (1 X 96 well plate)||---||In Stock||---|
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|Primary Cocktail||5535||500 µl||In-Cell Western Compatible||1:10||Human
|Detection Cocktail||5531||500 µl||In-Cell Western Compatible||1:10||N/A|
|Kit Analytes||Detection Dye||Ex(max) (nm)||Em(max) (nm)|
|Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)||DyLight® 800||777||794|
|Total p44/42 MAPK (Erk1/2)||DyLight® 680||692||712|
PhosphoPlus® p44/42 MAPK (Erk1/2) (Thr202/Tyr204) In-Cell Duet from Cell Signaling Technology (CST) provides an easy method to assess protein activation status using a multi-well plate scanner with near infrared detection capabilities, such as the LI-COR® Biosciences Odyssey® Infrared Imaging System. This kit contains a pre-optimized activation-state and total protein antibody cocktail, selected based on superior performance. Phosphorylated and total protein are detected simultaneously in the same well, allowing levels of phosphorylated protein to be normalized to total protein. A near infrared detection cocktail is also included.
Specificity / Sensitivity
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) detects endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. This antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases. Total p44/42 MAPK detects endogenous levels of total p44/42 MAP kinase (Erk1/Erk2) protein. In some systems this antibody may recognize p42/Erk2 more readily than p44/Erk1. The antibody does not cross-react with JNK/SAPK or p38 MAP kinase.
Analysis of HT-1080 cells exposed to varying concentrations of U0126 (MEK1/2 Kinase Inhibitor) #9903 for 2 hours, followed by stimulation with TPA #4174 for 30 minutes. The phosphorylation status of p44/42 MAPK (Erk1/2), as well as the total protein expression level, was measured simultaneously using the PhosphoPlus® p44/42 MAPK (Erk1/2) (Thr202/Tyr204) In-Cell Duet (ICW Compatible) #7263. With increasing concentrations of U0126, a significant decrease (~10-fold) in phospho-Erk signal as compared to the TPA-stimulated control was observed, while total Erk protein levels remained unchanged and were used to normalize the data. When using phospho-Erk as a measurement, the IC50 of this compound was 1 μM. Data and images were generated using the LI-COR® Biosciences Odyssey® Infrared Imaging System.
Source / Purification
Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase or to the sequence of p42 MAP kinase.
Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.
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For Research Use Only. Not For Use In Diagnostic Procedures.
LI-COR® is a registered trademark of LI-COR, Inc.
Odyssey® is a registered trademark of LI-COR, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.