FlowCellect™ EGFR RTK Activation Dual Detection Kit

FlowCellect™ EGFR RTK Activation Dual Detection Kit

FlowCellect

25 Tests / Kit

EGF pathway activation through EGFR plays a key role in the regulation of essential cellular processes, and abnormality of EGF signaling is linked to cancer.

Millipore’s FlowCellect™ EGFR RTK Activation Dual Detection kit includes two directly conjugated antibodies, a phospho-specific Anti-phospho-EGFR (Tyr1173)-Alexa Fluor® 488 and an Anti-EGFR-PerCP conjugated antibody to measure total levels of EGFR. This two color flow cytometry kit is designed to detect the extent of EGF pathway activation by measuring the EGFR phosphorylation in relative to the total EGFR expression in any given cell population. By doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of EGFR activation after stimulation. Moreover, simultaneous measurement of both total and phospho-EGFR confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho:total ratio within a mixed population.

All FlowCellect kits are supplied with optimized fixation, permeabilization, wash, and assay buffers to provide researchers the ability to detect the activation of EGFR in flow applications. Sufficient reagents are provided for 25 two-color samples. The kit includes all optimized fluorescently labeled antibodies and buffers necessary for cell preparation and analysis.

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). Mutations affecting EGFR expression or activity could result in cancer.

EGFR exists on the cell surface and is activated by binding of its specific ligands, including EGF and transforming growth factor α (TGFα). Upon activation, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. EGFR may also pair with another member of the ErbB receptor family, such as ErbB2/Her2/neu, to create an activated heterodimer. There is also evidence to suggest that activated EGFRs form clusters, although it remains unclear whether this clustering is important for activation itself or occurs subsequent to activation of individual dimers.

EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. As a result, autophosphorylation of five tyrosine (Y) residues in the C-terminal domain of EGFR occurs. These are Y992, Y1045, Y1068, Y1148 and Y1173. This autophosphorylation elicits downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their own phosphotyrosine-binding SH2 domains. These downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation. Such proteins modulate phenotypes such as cell migration, adhesion, and proliferation. The kinase domain of EGFR can also cross-phosphorylate tyrosine residues of other receptors it is aggregated with, and can itself be activated in that manner.

Flow Cytometry

Human

• Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
• 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.

4-8°C for antibodies and buffers

FlowCellect™ EGFR RTK Activation Dual Detection Kit

Flow Cellect

ExpressPro

Monoclonal Antibody

Research

Lot to lot in-process testing on all kit components

• 1. 20X Anti-phospho-EGFR (Tyr1173) AlexaFluor® 488 Antibody: (Part No. CS204825) One vial containing 150 µL.
• 2. 20X Anti-EGFR-PerCP Conjugated Monoclonal Antibody: (Part No. CS205071) One vial containing 150 µL.
• 3. Fixation Buffer: (Part No. CS202122) One bottle containing 13 mL.
• 4. 10X Wash Buffer: (Part No. CS202123) One bottle containing 13 mL.
• 5. 5X Assay Buffer: (Part No. CS202124) One 55 mL bottle
• 6. 1X Permeabilization Buffer: (Part No. CS203284) One bottle containing 13 mL.

Mouse

Fluorescent