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Confocal immunofluorescent analysis of paraffin-embedded H1975, H3255, HCC827, and H1650 xenografts using PathScan® EGF Receptor Activation Multiplex IF Kit. Red = EGF receptor, green = phospho-EGF receptor (Tyr1068), and blue pseudocolor = phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204).

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Confocal immunofluorescent analysis of serum-starved A431 cells, treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml) as indicated, using PathScan® EGF Receptor Activation Multiplex IF Kit. Red = EGF receptor, green = phospho-EGF receptor (Tyr1068), and blue pseudocolor = phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204).

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Immunofluorescence (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade, 100% and 95%.
  3. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  4. Antigen Unmasking EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
  5. 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
  6. 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.
  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Paraffin Sections (IF-P)

NOTE: Do not allow slides to dry at any time during this procedure.

Deparaffinization/Rehydration:

  1. Incubate sections in three washes of xylene for 5 minutes each.
  2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
  3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  4. Rinse sections twice in dH2O for 5 minutes each.

Antigen Unmasking:

NOTE: Consult product datasheet for specific recommendation for the unmasking solution.

  1. For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0: follow with 15 min at a sub-boiling temperature. No cooling is necessary.

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. Rinse three times in 1X PBS for 5 minutes each.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary cocktail.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  8. Incubate 1–2 hours at room temperature in the dark.
  9. inse three times in 1X PBS for 5 minutes each.
  10. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), or Prolong® Gold AntiFade Reagent with DAPI (#8961).
  11. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

protocol id: 444

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 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, 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, and then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes 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 minutes.
  2. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary cocktail.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  7. Incubate 1–2 hours at room temperature in the dark.
  8. Rinse three times in 1X PBS for 5 minutes each.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), or Prolong® Gold Antifade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

protocol id: 424

Product Description

The PathScan® EGF Receptor Activation Multiplex IF Kit offers a novel method to simultaneously monitor the expression, localization, and activation state of EGF receptor, as well as downstream signaling through Erk1/2, using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. This kit contains a cocktail of three high quality primary antibodies targeted against total EGF receptor, phospho-EGF receptor (Tyr1068), and phospho-p44/42 (Erk1/2) (Thr202/Tyr204), as well as a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody and dye pairings have been pre-optimized, and each kit contains enough reagents for 100 assays (based on a working volume of 100 μl/test).


Specificity / Sensitivity

EGF receptor antibody detects endogenous levels of total EGF receptor protein and does not cross-react with other ErbB family members. Phospho-EGF receptor (Tyr1068) antibody detects endogenous EGF receptor only when phosphorylated at Tyr1068. This antibody may cross-react weakly with other tyrosine-phosphorylated proteins. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody detects endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. This antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases.


Species Reactivity: Human, Monkey

Source / Purification

Monoclonal antibodies were produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase and Tyr1068 of human EGF receptor, or with a fusion protein containing the cytoplasmic domain of human EGF receptor.

The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane receptor tyrosine kinase that belongs to the HER/ErbB protein family that includes HER2/ErbB2/neu, HER3/ErbB3, and HER4/ErbB4 (1,2). Ligand binding results in receptor homo- and heterodimerization which stimulates its intrinsic tyrosine kinase activity. Autophosphorylation at residues Tyr992, Tyr1045, Tyr1068, Tyr1148, and Tyr1173, as well as c-Src mediated phosphorylation at Tyr845 and Tyr1101 promote docking of SH2 domain-bearing signaling proteins resulting in cellular responses to EGFR activation (2-6). For example, phosphorylation of Tyr1068 facilitates recruitment of Grb2 which, via association with Sos, stimulates the GTP binding activity of Ras, leading to the activation of MAP kinase and other signaling cascades (7). Following activation, EGFR is rapidly endocytosed and either recycled back to the plasma membrane or targeted for lysosomal degradation (8). Dysregulation of EGFR signaling through activating mutations or gene amplification has been implicated in the pathogenesis of many human malignancies, leading to intense clinical study of this pathway (9-13).


1.  Yarden, Y. and Sliwkowski, M.X. (2001) Nature Rev. Mol. Cell. Biol. 2, 127-137.

2.  Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.

3.  Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.

4.  Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.

5.  Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.

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.  Lai, W.H. et al. (1989) J Cell Biol 109, 2741-9.

9.  Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.

10.  Press, M.F. and Lenz, H.J. (2007) Drugs 67, 2045-75.

11.  Baselga, J. (2002) Oncologist 7 Suppl 4, 2-8.

12.  Foon, K.A. et al. (2004) Int J Radiat Oncol Biol Phys 58, 984-90.

13.  Sridhar, S.S. et al. (2003) Lancet Oncol 4, 397-406.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PathScan® is a trademark of Cell Signaling Technology, Inc.