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Typical phospho-EGFR (Tyr 1173) Standard Curve 100 µL of progressive 2 fold dilutions of the full length EGFR standard was run as described in ...
Phospho-EGFR (Tyr1173) STAR ELISA Assay kit
Description:
Phospho-EGFR (Tyr1173) STAR ELISA Kit
Trade Name:
Upstate (Millipore)
Qty/Pk:
96 assays
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.
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.
Key Applications:
ELISA
Species Reactivity:
- Human
- Mouse
- Rat
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.
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."
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."
Brand Family:
Upstate
Sensitivity:
Sensitivity: 1 unit/mL.
Range of Detection: 1.6 to 100 units/mL
Range of Detection: 1.6 to 100 units/mL
Storage Conditions:
1 year at 4°C
Promotional Text:
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Modifications:
Phosphorylation
Gene Symbol:
- EGFR
- ERBB1
- ERBB
- mENA
Product Name:
Phospho-EGFR (Tyr1173) STAR ELISA Assay kit
Alternate Names:
- ERBB, ERBB1, epidermal growth factor receptor
- EGF Receptor
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
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
Analytes Available:
EGFR
Quality Assurance:
Routinely evaluated by ELISA.
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.
UniProt Number:
Detection Methods:
- Chromogenic
- Chromogenic
Entrez Gene Number: