FlowCellect™ EGFR/MAPK Pathway Activation Detection Kit

FlowCellect™ EGFR/MAPK Pathway Activation Detection Kit

FlowCellect

25 Tests / Kit

Millipore’s FlowCellect™ EGFR/MAPK Activation Detection kit includes two directly conjugated phospho specific antibodies: pEGFR(Tyr1173)-Alexa Fluor 488 and pERK (Thr202/Tyr204, Thr185/Tyr187)-PE, along with optimized fixation, permeabilization, wash and assay buffers to provide researchers the ability to detect the activation of EGFR and ERK at the receptor and downstream signaling level.
The ability to detect the phosphorylation of EGFR and ERK simultaneously allows researchers to profile EGF mediated MAPK activation within a cell type of interest. Alterations in these signaling profiles can be used to monitor the effect of a particular upstream treatment targeting the pathway.
Antibodies against phospho-EGFR and phospho-ERK1/2 are provided in the kit to measure the extent of each proteins activation state. A431 cells were used as a model system to demonstrate EGF mediated activation of EGFR as well as its’ downstream effector kinase ERK. The treated cells were compared to untreated cells to verify the detection of both activated proteins by flow cytometry. When performing a dual color stain, the majority of the treated cells are double positive indicating activation of MAPK through EGFR.
Sufficient reagents are provided for 25 3-color samples. The kit includes all optimized fluorescently labeled antibodies and buffers necessary for cell preparation and analysis.

The Epidermal Growth Factor Receptor (EGFR) is one of the Receptor Tyrosine Kinases which can activate the MAPK pathway, one of three major signaling pathways downstream of EGFR. EGFR belongs to ErbB receptor superfamily, a group of transmembrane glycoprotein receptors, consisting of four members: EGFR (ErbB-1), ErbB-2 (c-neu, HER2), ErbB-3 (HER3), and ErbB-4 (HER 4).

Critical cellular decisions such as proliferation, migration, and differentiation, are regulated by
stimulatory cues from the extracellular environment, primarily growth factors, extracellular
matrix (ECM) proteins, and adhesion molecules presented on the surface of neighboring cells. Once these cues are received, cells undergo complex signaling cascades such as the Mitogen Activated Protein Kinase (MAPK) pathway in order to respond to their environmental stimuli.

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from
their receptors to regulate gene expression and prevent apoptosis. Once activated by phosphorylation Extra-cellular signal Related Kinase (ERK) enters the nucleus and activates target proteins associated with cellular proliferation and migration.

Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Due to the complexity of the pathway and its involvement in many malignancies the EGFR/MAPK pathway is studied by numerous researchers looking for insight into the mechanisms involved in the formation and progression of cancers.

Flow Cytometry

Human

• Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
• Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

Lot to lot in-process testing on all kit components

• 1. 20X Anti-phospho-EGFR (Tyr1173), clone 9H2; Alexa Fluor 488 Conjugate: (Part No. CS204825) One vial containing 150 mL antibody.
• 2. 20X Anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), clone AW39R; R-Phycoerythrin Conjugate: (Part No. CS203329) One vial containing 150 mL antibody.
• 3. Fixation Buffer: (Part No. CS202122) One bottle containing 13 mL buffer.
• 4. 10X Wash Buffer: (Part No. CS202123) One bottle containing 13 mL buffer.
• 5. 5X Assay Buffer: (Part No. CS202124) One bottle containing 55 mL buffer.
• 6. 1X Permeabilization Buffer: (Part No. CS202125) One bottle containing 13 mL buffer.

Mouse

4-8°C for antibodies and buffers

Fluorescent

FlowCellect™ EGFR/MAPK Pathway Activation Detection Kit

Flow Cellect

Monoclonal Antibody

Research