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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of NCI-H1650 cells (blue) and NCI-H3255 cells (green) using EGF Receptor (L858R Mutant Specific) (43B2) Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 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. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  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 antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

EGFR (L858R Mutant Specific) (43B2) Rabbit mAb (PE Conjugate) detects endogenous levels of EGFR mutant L858R protein. The antibody may cross-react with wildtype EGFR and other HER family members when highly over-expressed. Careful titration of this antibody may be required to obtain optimal specificity.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to L858R mutant sequence of human EGF receptor.

Product Description

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 (L858R Mutant Specific) (43B2) Rabbit mAb #3197.


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 USA 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.


Entrez-Gene Id 1956
Swiss-Prot Acc. P00533


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.
U.S. Patent No. 5,675,063.

64716
EGF Receptor (L858R Mutant Specific) (43B2) Rabbit mAb (PE Conjugate)