Enzyme Solution (12 ml)

Analytes Available	Species Reactivity	Key Applications	Detection Methods
EGFR	H, M, R	ELISA	Chromogenic

Analytes Available

Species Reactivity

Key Applications

Detection Methods

EGFR

H, M, R

ELISA

Chromogenic

Description
Catalogue Number	17-461
Brand Family	Upstate
Trade Name	STAR Upstate
Description	Phospho-EGFR (Tyr1173) STAR ELISA Kit
Alternate Names	Epidermal growth factor receptor Proto-oncogene c-ErbB-1 Receptor tyrosine-protein kinase erbB-1 EGF Receptor EGFR ERBB ERBB1
Background Information	I. TEST PRINCIPLE The UPSTATE colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The EGFR plate is coated with a specific mouse monoclonal anti-EGFR capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing EGFR antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-EGFR antibody to detect the captured phospho-EGFR (Tyr1173) on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard curve. II. EGFR BACKGROUND Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a key role in the regulation of essential normal cellular processes and in the pathophysiology of hyperproliferative diseases such as cancer. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase known to be essential for mediation of both proliferative and survival signals to cells. Activation of the EGFR signaling pathway has been linked with increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. EGFR is found in most solid tumors and its autophosphorylation activates the intrinsic kinase activity toward heterologous substrates, as well as creating docking sites for adapter proteins for multiple adapter proteins to bind and nucleate signaling complexes that activate the Ras, PI3 Kinase, and PLC pathways. Many of the EGFR mutations are directly related to various diseases and are the target of numerous drugs. For instance, the amino acid substitution L858R is one of several heterozygous mutations that have been identified in Non-Small-Cell Lung Cancer (NSCLC) patients who have clinical responses to the EGFR inhibitor Iressa®. There is some evidence that these mutations result in elevated activity and enhanced sensitivity to Iressa®. In patients with tumors bearing Iressa®-sensitive mutations, resistant subclones containing an additional EGFR mutation, T790M, emerge in the presence of the drug. It has been shown experimentally that the T790M mutation leads to high-level functional resistance to Iressa. In patients with tumors bearing Iressa®-sensitive mutations (eg. L858R, L861Q), resistant subclones containing the T790M mutation emerge in the presence of the drug.
Materials Required but Not Delivered	1. Multi-channel or repeating pipettes 2. Plate shaker (optional) 3. Pipettors & tips capable of accurately measuring 1-1000 %micro;L 4. Graduated serological pipettes 5. 96-well microtiter Plate Reader with 450 nm filter 6. Graphing software for plotting data or graph paper for manual plotting of data 7. Microfuge tubes for standard and sample dilutions 8. Mechanical vortex 9. 1 liter container 10. Distilled or deionized water

Description

Catalogue Number

17-461

Brand Family

Upstate

Trade Name

STAR
Upstate

Description

Phospho-EGFR (Tyr1173) STAR ELISA Kit

Alternate Names

Epidermal growth factor receptor
Proto-oncogene c-ErbB-1
Receptor tyrosine-protein kinase erbB-1
EGF Receptor
EGFR
ERBB
ERBB1

Background Information

I. TEST PRINCIPLE
The UPSTATE colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The EGFR plate is coated with a specific mouse monoclonal anti-EGFR capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing EGFR antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-EGFR antibody to detect the captured phospho-EGFR (Tyr1173) on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard curve.

II. EGFR BACKGROUND

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a key role in the regulation of essential normal cellular processes and in the pathophysiology of hyperproliferative diseases such as cancer. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase known to be essential for mediation of both proliferative and survival signals to cells. Activation of the EGFR signaling pathway has been linked with increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. EGFR is found in most solid tumors and its autophosphorylation activates the intrinsic kinase activity toward heterologous substrates, as well as creating docking sites for adapter proteins for multiple adapter proteins to bind and nucleate signaling complexes that activate the Ras, PI3 Kinase, and PLC pathways.
Many of the EGFR mutations are directly related to various diseases and are the target of numerous drugs. For instance, the amino acid substitution L858R is one of several heterozygous mutations that have been identified in Non-Small-Cell Lung Cancer (NSCLC) patients who have clinical responses to the EGFR inhibitor Iressa®. There is some evidence that these mutations result in elevated activity and enhanced sensitivity to Iressa®. In patients with tumors bearing Iressa®-sensitive mutations, resistant subclones containing an additional EGFR mutation, T790M, emerge in the presence of the drug. It has been shown experimentally that the T790M mutation leads to high-level functional resistance to Iressa. In patients with tumors bearing Iressa®-sensitive mutations (eg. L858R, L861Q), resistant subclones containing the T790M mutation emerge in the presence of the drug.

Materials Required but Not Delivered

1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 %micro;L
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water

Product Information
Components	1. Capture Plate pre-coated with anti-human EGFR antibody: (Part No. 17-461A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch. 2. Anti-EGFR (Tyr1173) detection antibody: (Part No. 17-461B) One bottle (11 mL) of anti-EGFR (Tyr1173) detection antibody containing sodium azide, ready to use. 3. ELISA Diluent: (Part No. 17-461C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use. 4. 25X ELISA Wash Buffer: (Part No. 17-461D) One bottle (50 mL) of 25X ELISA Wash Buffer. 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-461E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosol. 6. HRP Diluent: (Part No. 17-461F) One bottle (25 mL) of HRP Diluent containing thimerosol. 7.TMB Solution: (Part No. 17-461G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use. 8. Stop Solution: (Part No. 17-461H) One bottle (25 mL) of stop solution, ready to use. 9.EGFR (Tyr1173) Standard: (Part No. 17-461I) Two vials of EGFR (Tyr1173) standard, lyophilized. 10.Plate Covers: Two plate covers.
Detection method	Chromogenic

Product Information

Components

1. Capture Plate pre-coated with anti-human EGFR antibody: (Part No. 17-461A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
2. Anti-EGFR (Tyr1173) detection antibody: (Part No. 17-461B) One bottle (11 mL) of anti-EGFR (Tyr1173) detection antibody containing sodium azide, ready to use.
3. ELISA Diluent: (Part No. 17-461C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
4. 25X ELISA Wash Buffer: (Part No. 17-461D) One bottle (50 mL) of 25X ELISA Wash Buffer.
5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-461E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosol.
6. HRP Diluent: (Part No. 17-461F) One bottle (25 mL) of HRP Diluent containing thimerosol.
7.TMB Solution: (Part No. 17-461G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
8. Stop Solution: (Part No. 17-461H) One bottle (25 mL) of stop solution, ready to use.
9.EGFR (Tyr1173) Standard: (Part No. 17-461I) Two vials of EGFR (Tyr1173) standard, lyophilized.
10.Plate Covers: Two plate covers.

Detection method

Chromogenic

Applications
Key Applications	ELISA

Applications

Key Applications

ELISA

Biological Information
Species Reactivity	Human Mouse Rat
Analytes Available	EGFR
Entrez Gene Number	NM_005228.3 NM_201282.1 NM_201283.1 NM_201284.1
Gene Symbol	EGFR ERBB1 ERBB mENA
Modifications	Phosphorylation
UniProt Number	P00533
UniProt Summary	FUNCTION: SwissProt: P00533 # Isoform 2/truncated isoform may act as an antagonist. SIZE: 1210 amino acids; 134277 Da SUBUNIT: Binds RIPK1. CBL interacts with the autophosphorylated C- terminal tail of the EGF receptor. Part of a complex with ERBB2 and either PIK3C2A or PIK3C2B. The autophosphorylated form interacts with PIK3C2B, maybe indirectly. Interacts with PELP1. SUBCELLULAR LOCATION: Cell membrane; Single-pass type I membrane protein. & Isoform 2: Secreted. TISSUE SPECIFICITY: Expressed in placenta. Isoform 2 is also expressed in ovarian cancers. PTM: Phosphorylation of Ser-695 is partial and occurs only if Thr- 693 is phosphorylated. & Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occur.DISEASE:SwissProt: P00533 # Defects in EGFR are associated with lung cancer [MIM:211980]. SIMILARITY: SwissProt: P00533 ## Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily. & Contains 1 protein kinase domain. MISCELLANEOUS: Binding of EGF to the receptor leads to dimerization, internalization of the EGF-receptor complex, induction of the tyrosine kinase activity, stimulation of cell DNA synthesis, and cell proliferation.

Biological Information

Species Reactivity

Human
Mouse
Rat

Analytes Available

EGFR

Entrez Gene Number

Gene Symbol

EGFR
ERBB1
ERBB
mENA

Modifications

Phosphorylation

UniProt Number

P00533

UniProt Summary

FUNCTION: SwissProt: P00533 # Isoform 2/truncated isoform may act as an antagonist.
SIZE: 1210 amino acids; 134277 Da
SUBUNIT: Binds RIPK1. CBL interacts with the autophosphorylated C- terminal tail of the EGF receptor. Part of a complex with ERBB2 and either PIK3C2A or PIK3C2B. The autophosphorylated form interacts with PIK3C2B, maybe indirectly. Interacts with PELP1.
SUBCELLULAR LOCATION: Cell membrane; Single-pass type I membrane protein. & Isoform 2: Secreted.
TISSUE SPECIFICITY: Expressed in placenta. Isoform 2 is also expressed in ovarian cancers.
PTM: Phosphorylation of Ser-695 is partial and occurs only if Thr- 693 is phosphorylated. & Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occur.DISEASE:SwissProt: P00533 # Defects in EGFR are associated with lung cancer [MIM:211980].
SIMILARITY: SwissProt: P00533 ## Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily. & Contains 1 protein kinase domain.
MISCELLANEOUS: Binding of EGF to the receptor leads to dimerization, internalization of the EGF-receptor complex, induction of the tyrosine kinase activity, stimulation of cell DNA synthesis, and cell proliferation.

Physicochemical Information
Sensitivity	Sensitivity: 1 unit/mL. Range of Detection: 1.6 to 100 units/mL

Physicochemical Information

Sensitivity

Sensitivity: 1 unit/mL.
Range of Detection: 1.6 to 100 units/mL

Product Usage Statements
Quality Assurance	Routinely evaluated by ELISA.
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Quality Assurance

Routinely evaluated by ELISA.

Usage Statement

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	1 year at 4°C

Storage and Shipping Information

Storage Conditions

1 year at 4°C

Packaging Information
Material Size	96 assays

Packaging Information

Material Size

96 assays

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

办公用品

Enzyme Solution (12 ml)

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