EGFR (Phospho-Thr693)Colorimetric Cell-Based ELISA Kit (CBCAB00135)

Product Name:	EGFR (Phospho-Thr693)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00135
ELISA Type:	Cell-Based
Target:	EGFR (Phospho-Thr693)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The EGFR (Phospho-Thr693) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect EGFR protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated EGFR in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on EGFR phosphorylation.

Qualitative determination of EGFR (Phospho-Thr693) concentration is achieved by an indirect ELISA format. In essence, EGFR (Phospho-Thr693) is captured by EGFR (Phospho-Thr693)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 1956, UniProt ID: P00533, OMIM: 131550/211980, Unigene: Hs.488293
Gene Symbol:	EGFR
Sub Type:	Phospho

UniProt Protein Function:	EGFR: a receptor tyrosine kinase. This is a receptor for epidermal growth factor (EGF) and related growth factors including TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30, and vaccinia virus growth factor. EGFR is involved in the control of cell growth and differentiation. It is a single-pass transmembrane tyrosine kinase. Ligand binding to this receptor results in receptor dimerization, autophosphorylation (in trans), activation of various downstream signaling molecules and lysosomal degradation. It can be phosphorylated and activated by Src. Activated EGFR binds the SH2 domain of phospholipase C-gamma (PLC-gamma), activating PLC-gamma-mediated downstream signaling. Phosphorylated EGFR binds Cbl, leading to its ubiquitination and degradation. Grb2 and SHC bind to phospho-EGFR and are involved in the activation of MAP kinase signaling pathways. Phosphorylation on Ser and Thr residues is thought to represent a mechanism for attenuation of EGFR kinase activity. EGFR is overexpressed in breast, head and neck cancers, correlating with poor survival. Activating somatic mutations are seen in lung cancer, corresponding to the minority of patients with strong responses to the EGFR inhibitor Iressa (gefitinib). Mutations and amplifications are also seen in glioblastoma, and upregulation is seen in colon cancer and neoplasms. In xenografts, inhibitors synergize with cytotoxic drugs in the inhibition of many tumor types. Inhibitors include: Iressa/ZD1839, Erbitux, Tarceva, and lapatinib. Four alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Tumor suppressor; Protein kinase, tyrosine (receptor); Protein kinase, TK; Kinase, protein; Membrane protein, integral; EC 2.7.10.1; TK group; EGFR family Chromosomal Location of Human Ortholog: 7p12 Cellular Component: extracellular space; endoplasmic reticulum membrane; nuclear membrane; cell surface; focal adhesion; basolateral plasma membrane; integral to membrane; lipid raft; Golgi membrane; membrane; perinuclear region of cytoplasm; cytoplasm; apical plasma membrane; plasma membrane; AP-2 adaptor complex; endosome membrane; nucleus; receptor complex; endosome Molecular Function:identical protein binding; epidermal growth factor receptor activity; epidermal growth factor binding; nitric-oxide synthase regulator activity; transmembrane receptor protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; protein phosphatase binding; protein kinase binding; actin filament binding; integrin binding; protein binding; transmembrane receptor activity; enzyme binding; MAP kinase kinase kinase activity; protein heterodimerization activity; ubiquitin protein ligase binding; protein-tyrosine kinase activity; double-stranded DNA binding; chromatin binding; glycoprotein binding; ATP binding Biological Process: circadian rhythm; diterpenoid metabolic process; positive regulation of nitric oxide biosynthetic process; nerve growth factor receptor signaling pathway; activation of MAPKK activity; alkanesulfonate metabolic process; protein insertion into membrane; positive regulation of vasodilation; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; positive regulation of MAP kinase activity; positive regulation of fibroblast proliferation; cell-cell adhesion; ovulation cycle; cell surface receptor linked signal transduction; hair follicle development; positive regulation of superoxide release; negative regulation of mitotic cell cycle; positive regulation of DNA repair; fibroblast growth factor receptor signaling pathway; digestive tract morphogenesis; response to osmotic stress; phospholipase C activation; response to hydroxyisoflavone; hydrogen peroxide metabolic process; positive regulation of transcription from RNA polymerase II promoter; response to oxidative stress; regulation of nitric-oxide synthase activity; response to calcium ion; negative regulation of protein catabolic process; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; negative regulation of epidermal growth factor receptor signaling pathway; axon guidance; tongue development; embryonic placenta development; peptidyl-tyrosine phosphorylation; translation; protein amino acid autophosphorylation; positive regulation of smooth muscle cell proliferation; signal transduction; positive regulation of synaptic transmission, glutamatergic; learning and/or memory; positive regulation of cell proliferation; salivary gland morphogenesis; response to stress; regulation of peptidyl-tyrosine phosphorylation; epidermal growth factor receptor signaling pathway; ossification; phosphoinositide-mediated signaling; MAPKKK cascade; liver development; cell proliferation; positive regulation of protein kinase B signaling cascade; cerebral cortex cell migration; calcium-dependent phospholipase A2 activation; positive regulation of vasoconstriction; innate immune response; positive regulation of protein amino acid phosphorylation; astrocyte activation; positive regulation of DNA replication; positive regulation of phosphorylation; response to cobalamin; positive regulation of cell migration; lung development; positive regulation of inflammatory response Disease: Lung Cancer
NCBI Summary:	The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. Mutations in this gene are associated with lung cancer. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2010]
UniProt Code:	P00533
NCBI GenInfo Identifier:	2811086
NCBI Gene ID:	1956
NCBI Accession:	P00533.2
UniProt Secondary Accession:	P00533,O00688, O00732, P06268, Q14225, Q68GS5, Q92795 Q9BZS2, Q9GZX1, Q9H2C9, Q9H3C9, Q9UMD7,
UniProt Related Accession:	P00533
Molecular Weight:	69,228 Da
NCBI Full Name:	Epidermal growth factor receptor
NCBI Synonym Full Names:	epidermal growth factor receptor
NCBI Official Symbol:	EGFR
NCBI Official Synonym Symbols:	ERBB; HER1; mENA; ERBB1; PIG61; NISBD2
NCBI Protein Information:	epidermal growth factor receptor; proto-oncogene c-ErbB-1; cell growth inhibiting protein 40; cell proliferation-inducing protein 61; receptor tyrosine-protein kinase erbB-1; avian erythroblastic leukemia viral (v-erb-b) oncogene homolog
UniProt Protein Name:	Epidermal growth factor receptor
UniProt Synonym Protein Names:	Proto-oncogene c-ErbB-1; Receptor tyrosine-protein kinase erbB-1
Protein Family:	Pro-epidermal growth factor
UniProt Gene Name:	EGFR
UniProt Entry Name:	EGFR_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-EGFR (Phospho-Thr693) Antibody, Anti-EGFR Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)