Product Pathways - Tyrosine Kinase / Adaptors

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PathScan^® EGF Receptor Activation Multiplex IF Kit #7967

Products Included	Product No.	Volume	Applicaton	Dilution	Reactivity
Primary Cocktail	7972	100 µl	IF-IC, IF-P*	1:100	H, Mk, (M)
Detection Cocktail	7973	100 µl	IF-IC, IF-P*	1:100	N/A

Kit Analytes	Detection Dye	Ex(max) (nm)	Em(max) (nm)
EGF Receptor	Alexa Fluor® 555	555	565
Phospho-EGF Receptor (Tyr1068)	Alexa Fluor® 488	495	519
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)	Alexa Fluor® 647	650	665

Applications Key:  IF-P=Immunofluorescence (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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.

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.

Background

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. 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. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
5. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
6. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
7. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
8. Lai, W.H. et al. (1989) J Cell Biol 109, 2741-9.
9. Yarden, Y. and Sliwkowski, M.X. (2001) Nat Rev Mol Cell Biol 2, 127-37.
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.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

7967:

Immunofluorescence*

7972:

Immunofluorescence*

7973:

Immunofluorescence*

* Product-specific protocol.

Companion Products

• 4083 DAPI
• 4082 Hoechst 33342

*IF-P protocol recommended unmasking buffer: EDTAThe transfer of the secondary cocktail contained in this kit is contingent on the buyer using the purchased product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity), for immunocytochemistry, high content screening (HCS) analysis, or flow cytometry applications. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components 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 Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.

com. Alexa Fluor^® is a registered trademark of Molecular Probes, Inc.

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For Research Use Only. Not For Use In Diagnostic Procedures.