Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) Dual Detect CELISA Assay Kit (Fluorogenic Detection)
Description:
Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) Dual Detect CELISA Assay Kit (Fluorogenic Detection)
Trade Name:
Dual Detect CELISA
Qty/Pk:
96 Assays
Product Overview:
Dual Detect CELISA is a straightforward and efficient cell-based ELISA assay that eliminates the need to prepare cell lysates. It allows for the measurement of the relative expression levels of both the total and the phosphorylated forms of the target protein in whole cells in a convenient 96 well microplate format using dual fluorescent detection. This allows for the normalization of total protein between wells, thus allowing a more accurate measurement of phosphorylation levels among cell and/or growth condition differentials.
Cells are cultured directly in the microplate and stimulated as desired. They are then fixed and simultaneously incubated with two primary antibodies. One is a phosphospecific antibody directed against the phosphorylated epitope of the protein that correlates with it activation and the other is a total antibody that recognizes the target-protein, regardless of phosphorylation status. the levels of both the total target protein and the phosphorylated protein are measured simultaneously in a single well using a double immunoenzymatic labeling procedure, either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for HRP and AP. The fluorescence of the phosphorylated protein is normalized to the total protein in each well thus reducing well-to-well variations. The Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) kit contains the components required to run the CELISA using fluorogenic substrates to measure both total Erk 1/2 and phosphorylated Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) in the context of whole cells.
Cells are cultured directly in the microplate and stimulated as desired. They are then fixed and simultaneously incubated with two primary antibodies. One is a phosphospecific antibody directed against the phosphorylated epitope of the protein that correlates with it activation and the other is a total antibody that recognizes the target-protein, regardless of phosphorylation status. the levels of both the total target protein and the phosphorylated protein are measured simultaneously in a single well using a double immunoenzymatic labeling procedure, either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for HRP and AP. The fluorescence of the phosphorylated protein is normalized to the total protein in each well thus reducing well-to-well variations. The Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) kit contains the components required to run the CELISA using fluorogenic substrates to measure both total Erk 1/2 and phosphorylated Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) in the context of whole cells.
Background Information:
The classical, canonical MAPK signaling pathway is an evolutionarily conserved pathway that controls the growth and survival of a large group of human tumors where it is shown to have increased and sustainable activation. It is involved in the control of many fundamental cellular processes that include cell proliferation, survival, differentiation, apoptosis, motility and metabolism. It accomplishes this through the promotion of cell proliferation and preventing apoptosis. In a normal cell, the signaling pathway is initiated by the binding of a growth factor such as EGF, VEGF, PDGF, HGF, and FGF, to its respective receptor. This ligand binding activates the receptor tyrosine kinase (RTK) that results in its binding of Grb2 that binds to specific phosphorylated residues in the intracellular tail portion of the receptor. SOS binds to Grb2 to activate the small G-protein Ras by catalyzing the replacement of GDP to GTP. In its GTP-bound active state, Ras activates the kinase activity of the Ser/Thr kinase Raf (MAP Kinase Kinase Kinase). Raf then binds to and activates MEK (MAP Kinase Kinase) by phosphorylating it on the two residue motif. MEK, a dual kinase (a Ser/Thr and a Tyr kinase), phosphorylates and activates ERK on the TxY motif. The regulation of a large number of cellular processes are dependent upon the activation state of ERK, so the controlling of this pathway could have profound effects on various diseases. Many malignant cancers are characterized by the deregulation of the ERK signaling cascades. Cancerous cells do not respond to cell signaling that would normally result in cell cycle arrest or apoptosis (programmed cell death). In fact, constitutive ERK signaling contributes to the evolution of some of the most lethal forms of cancer. In some cancers, ERK is upregulated and results in the migration and invasion of the cancerous cells. Inhibition of the ERK expression reduces migration and invasion. Many tumors show an increase, sustained activation of this pathway. Cellular activities that MAPKs modulate include cell division, proliferation, survival, differentiation, apoptosis, motility and metabolism. This is one reason why the MAPK signaling pathway is a highly sought after anti-cancer drug target.
Species Reactivity:
- Human
- Mouse
- Rat
Quality Assurance:
Routinely QC tested
Components:
- 500X anti-Erk (Total) Antibody-15uL
- 500X anti-phospho Erk 1/2 (Thr202/Tyr204,Thr185/Tyr187) Antibody
- 500X Goat anti-Mouse HRP Detection Reagent
- 500X Goat anti-Rabbit AP Detection Reagent
- 200X HRP Substrate
- 200X AP Substrate
- Black Clear Bottom 96-well Cell Culture Plate
- 20% Tween® 20 (v/v)
- 20X TBS
- Substrate Diluent
UniProt Number:
Storage Conditions:
-20°C
Detection Methods:
Fluorescent
Modifications:
Phosphorylation
Product Name:
Erk 1/2 (Thr202/Tyr204)/(Thr185/Tyr187) Dual Detect CELISA Assay Kit (Fluorogenic Detection)
Entrez Gene Number:
Alternate Names:
- MAP Kinase
- p44/p42
Analytes Available:
Erk/MAPK 1/2