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54016
EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb (PE Conjugate) #54016

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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).

To Purchase # 54016S
Product # Size Price
54016S
100 µl  (50 tests) $ 364

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

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.

Product Usage Information

Application Dilution
Flow Cytometry 1:50

Storage:

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

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.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

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.

B. Fixation

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.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of EGFR E746-A750del mutant protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to E746-A750del mutant sequence of human EGFR.

Background

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.

  1. Lynch, T.J. et al. (2004) N Engl J Med 350, 2129-39.
  2. Pao W et al. (2004) Proc Natl Acad Sci U S A 101, 13306–11
  3. Haber, D.A. et al. (2005) Cold Spring Harb Symp Quant Biol 70, 419-26.
  4. Kosaka, T. et al. (2004) Cancer Res 64, 8919-23.
  5. Riely, G.J. et al. (2006) Clin Cancer Res 12, 7232-41.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.
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 busdev@cellsignal.com.