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8742
EGF Receptor (D38B1) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate)
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EGF Receptor (D38B1) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) #8742

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  1. IF

Immunofluorescent analysis of A549 cells, untreated (left) or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 15 min; +) (right), using EGF Receptor (D38B1) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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To Purchase # 8742S
Product # Size Price
8742S		 100 µl  (50 tests) $ 348

Custom Formulation

Supporting Data

REACTIVITY H M Mk
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 Alexa Fluor® 594 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated EGF Receptor (D38B1) XP® Rabbit mAb #4267.

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 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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Immunofluorescence (Immunocytochemistry)

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 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. 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.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

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.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 182

Specificity / Sensitivity

EGF Receptor (D38B1) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) recognizes 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

Source / Purification

Monoclonal antibody is produced by immunizing animals with a fusion protein containing the cytoplasmic domain of human EGF receptor.

Background

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).

  1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.
  2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.
  3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.
  4. Hubbard, S.R. et al. (1994) Nature 372, 746-54.
  5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.
  6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
  7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
  8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.
  9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
  10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

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.
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.