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EGF Receptor (D1D4J) XP® Rabbit mAb (Neutralizing) (PE Conjugate) #48685
Gallery: EGF Receptor (D1D4J) XP® Rabbit mAb (Neutralizing) (PE Conjugate) #48685
Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.
NOTE: If live cell staining is desired, proceed to Section C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
- Fix for 15 minutes at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells.
- If necessary, centrifuge to remove excess PBS.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 30-60 minutes at room temperature.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.
D. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted January 2009
revised June 2017
EGF Receptor (D1D4J) XP® Rabbit mAb (Neutralizing) (PE Conjugate) recognizes endogenous levels of total EGFR protein.Species Reactivity: Human, Mouse
Monoclonal antibody is produced by immunizing animals with mammalian cells expressing full length EGF receptor protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated EGFR (D1D4J) XP® Rabbit mAb (Neutralizing) #54359.
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.