Product: RayBio®
Human EGFR Phosphorylation Antibody Array 1 - For Simultaneously Detecting the Relative Levels of
Phosphorylation of EGF Receptors at 17 Different Phosphorylation
Sites
The EGFR family of membrane receptors consists of four
different proteins called EGFR/ErbB1/HER1, ErbB2/HER2,
ErbB3/HER3, and ErbB4/HER4. Under normal physiological
conditions, the ErbB receptors play crucial roles in
propagating signals regulating cell proliferation,
differentiation, motility, and apoptosis. EGF receptor family
shows clear differences between individual receptors, and also
a large overlap. ErbB1 is the family member with most
interaction partners and the highest percentage of tyrosine
residues with more than one binding partner. ErbB3 is
characterized by a large number of binding sites for
phosphatidylinositol-3-kinase (PI3K), while ErbB2 has only few
interaction partners with Shc as the most frequent one. ErbB1
and ErbB4 have a variety of phosphotyrosines that bind Grb2,
or Grb2 and Shc. The ErbB1 and ErbB4 have a greater diversity
of interaction partners than ErbB2 and ErbB3. ErbB1 and ErbB2
are often over-expressed or amplified in cancers, making them
important targets for drugs currently in use or under
development.
With the RayBio® Human EGF Phosphorylation Antibody Array 1,
researchers can now simultaneously detect the relative level
of phosphorylation of 17 different specific sites for Human
EGFR family in cell lysate. By monitoring the changes in
protein phosphorylation in your experimental model system, you
can verify pathway activation in your cell lines without
spending excess time and effort in performing an analysis of
immunoprecipitation and/or Western Blot.
By using RayBio® Human EGF Phosphorylation Antibody Array 1, treated
or untreated cell lysate is added into antibody array
membranes. The antibody array membranes are washed and
cocktail of biotin-congujated anti-EGFR is used to detect
phosphorylated EGFR on activated receptors. After incubation
with HRP-streptavidin, the signals are visualized by
chemiluminescence.
Fig. 1. Human epidermoid carcinoma cell
line, A431 cells that were 80-90% confluent were serum starved
overnight, then exposed to 100 ng/ml EGF for 20 minutes at 37
°C. Control cells were serum starved without the subsequent
stimulation with EGF. Cell lysates were prepared following the
"Preparation of Sample" portion of our protocol V.
To use the RayBio® Human EGF Phosphorylation Antibody Array 1, treated or
untreated cell lysate was added into antibody array
membrane. The antibody array membranes were washed and
cocktail of biotinylated anti-EGFR was used to detect
phosphorylated proteins on activated receptors. After
incubation with HRP-Conjugated Streptavidin, the signals were
visualized by chemiluminescence.
Fig. 2. Western blot analysis of extracts
from 100 ng/ml hEGF treated A431 cells and untreated
A431 cells. Phospho-EGFR (Tyr845) or Phospho- EGFR
(Tyr1173) antibodies was used in this assay.
Zhang J, Ji J-Y, Yu M,
Overholtzer M, et al. (2009). YAP-dependent induction of
amphiregulin identifies a non-cell-autonomous component
of the Hippo pathway. Nature Cell Biol. 11:1444-1450.
2
Holbro T, Hynes NE (2004).
ErbB receptors: directing key signaling networks
throughout life. Annu Rev Pharmacol Toxicol 44:
195–217
3
Normanno N, De Luca A, Bianco
C, Strizzi L, et al. Epidermal growth factor receptor (EGFR)
signaling in cancer. Gene. 2006:366(1):2-16.
4
Schulze WX, Deng L, Mann M.
Phosphotyrosine interactome of the ErbB-receptor kinase
family. (2005). Mol Systems Biol. 1:2005.0008 (doi:10.1038/msb4100012)
5
Huang RP, Huang R, Fan Y, and Lin Y
(2001). A novel method for high- throughput protein
profiling from conditioned media and patient’s sera..
Ana. Biochem. 294(1):55-62.
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