Confocal immunofluorescent analysis of A549 cells, serum-starved (left) or EGF-treated (#8916, right), using EGF Receptor (D28B1) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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This Cell Signaling Technology® antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated EGF Receptor (D38B1) XP® Rabbit mAb #4267.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
EGF Receptor (D38B1) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of total EGF receptor protein. The antibody does not cross-react with other proteins of the ErbB family. Species cross-reactivity by IF-IC is human only.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a fusion protein containing the cytoplasmic domain of human EGF receptor. The antibody was conjugated to Alexa Fluor® 555 under optimal conditions with an F/P ratio of 2-6.
The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
The manufacture, use, sale and import of this product is within the scope of one or more intellectual property rights (including patents and patent applications) owned or controlled by Cell Signaling Technology. The purchase of this product conveys to the buyer a non-transferrable right to use the purchased product only in research conducted by the buyer. The sale of the product is expressly conditioned on the buyer not using the products or its components (1) to analyze or reverse engineer the product for its chemical/physical properties and composition (including e.g., identification of the sequence); (2) in manufacturing; (3) to provide a service, information, or data to an unaffiliated third party for payment; (4) for therapeutic, diagnostic or prophylactic purposes; (5) resale, whether or not such product are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Cell Signaling Technology, Inc. Business Development at firstname.lastname@example.org.