Flow cytometric analysis of KYSE-450 cells (blue) and HCC827 cells (green) using EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb #2085.
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.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of EGFR E746-A750del mutant protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to E746-A750del mutant sequence of human EGFR.
The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Research studies have shown that somatic mutations in the tyrosine kinase domain of EGF receptor (EGFR) are present in a subset of lung adenocarinomas that respond to EGFR inhibitors, such as gefinitib and erlotinib (1-3). Two types of mutations account for approximately 90% of mutated cases: a specific point mutation, L858R, that occurs in exon 21 and short in-frame deletions in exon 19 (4,5). The most frequent exon 19 deletion is E746-A750, accounting for 90% of lesions at this site, although some rare variants occur.
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