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Typical phospho-Src (Tyr418) Standard Curve 100 µL of progressive 2 fold dilutions of the phospho-Src (Tyr418) standard included in the kit and...
phospho-Src (Tyr418) STAR ELISA Kit
Description:
phospho-Src (Tyr418) STAR ELISA Kit
Trade Name:
- STAR
- Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
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 Src plate is coated with a specific mouse monoclonal anti-Src 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 Src 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-Src antibody to detect the captured phospho-Src (Tyr418) 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.
Src Background
pp60c-Src is the prototype non-receptor tyrosine kinase. Homology to Src in three regions is shared by a number of proteins: SH1 being the catalytic domain, SH2 being a phospho-tyrosine binding domain, and SH3 being a proline-binding domain. The Src SH2 domain binds to phospho-Tyr527 (phosphorylated by Csk) within the Src molecule, causing Src to adopt an inactive conformation. Mutation of Tyr527 to phenylalanine removes the SH2-binding target and results in a constitutively active kinase. A second mutation at the conserved lysine residue at the ATP-binding site abolishes kinase activity, but without Tyr527, the conformation of the protein is open, allowing interaction with endogenous activators. Consequently, this double-mutant exhibits a dominant-negative phenotype.
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 Src plate is coated with a specific mouse monoclonal anti-Src 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 Src 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-Src antibody to detect the captured phospho-Src (Tyr418) 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.
Src Background
pp60c-Src is the prototype non-receptor tyrosine kinase. Homology to Src in three regions is shared by a number of proteins: SH1 being the catalytic domain, SH2 being a phospho-tyrosine binding domain, and SH3 being a proline-binding domain. The Src SH2 domain binds to phospho-Tyr527 (phosphorylated by Csk) within the Src molecule, causing Src to adopt an inactive conformation. Mutation of Tyr527 to phenylalanine removes the SH2-binding target and results in a constitutively active kinase. A second mutation at the conserved lysine residue at the ATP-binding site abolishes kinase activity, but without Tyr527, the conformation of the protein is open, allowing interaction with endogenous activators. Consequently, this double-mutant exhibits a dominant-negative phenotype.
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.
Entrez Gene Summary:
This gene is highly similar to the v-src gene of Rous sarcoma virus. This proto-oncogene may play a role in the regulation of embryonic development and cell growth. The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase. Mutations in this gene could be involved in the malignant progression of colon cancer. Two transcript variants encoding the same protein have been found for this gene.
UniProt Summary:
SIZE: 536 amino acids; 59835 Da
SUBUNIT: Interacts with DDEF1/ASAP1 via its SH3 domain. Interacts with CCPG1 (By similarity). Interacts with CDCP1, PELP1, TGFB1I1 and TOM1L2.
PTM: Phosphorylated on Tyr-530 by c-Src kinase (CSK). The phosphorylated form is termed pp60c-src. The phosphorylated tail interacts with the SH2 domain thereby repressing kinase activity.
SIMILARITY: SwissProt: P12931 ## Belongs to the protein kinase superfamily. Tyr protein kinase family. SRC subfamily. & Contains 1 protein kinase domain. & Contains 1 SH2 domain. & Contains 1 SH3 domain.
SUBUNIT: Interacts with DDEF1/ASAP1 via its SH3 domain. Interacts with CCPG1 (By similarity). Interacts with CDCP1, PELP1, TGFB1I1 and TOM1L2.
PTM: Phosphorylated on Tyr-530 by c-Src kinase (CSK). The phosphorylated form is termed pp60c-src. The phosphorylated tail interacts with the SH2 domain thereby repressing kinase activity.
SIMILARITY: SwissProt: P12931 ## Belongs to the protein kinase superfamily. Tyr protein kinase family. SRC subfamily. & Contains 1 protein kinase domain. & Contains 1 SH2 domain. & Contains 1 SH3 domain.
Quality Assurance:
Routinely evaluated by ELISA.
Components:
- 1. Capture Plate pre-coated with anti-Src antibody: (Part No. 17-468A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
- 2. Anti-phospho-Src (Tyr418) detection antibody: (Part No. 17-468B) One bottle (11 mL) of anti- phospho-Src (Tyr418) detection antibody containing sodium azide, ready to use.
- 3. ELISA Diluent: (Part No. 17-468C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
- 4. 25X ELISA Wash Buffer: (Part No. 17-468D) One bottle (50 mL) of 25X ELISA Wash Buffer.
- 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-468E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosol.
- 6. HRP Diluent: (Part No. 17-468F) One bottle (25 mL) of HRP Diluent containing thimerosol.
- 7. TMB Solution: (Part No. 17-468G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
- 8. Stop Solution: (Part No. 17-468H) One bottle (25 mL) of stop solution, ready to use.
- 9. Phospho-Src (Tyr418) Standard: (Part No. 17-468I) Two vials of phospho-Src (Tyr418) standard, lyophilized.
- 10. Plate Covers: Two plate covers.
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
UniProt Number:
Storage Conditions:
1 year at 4°C
Detection Methods:
- Chromogenic
- Chromogenic
Modifications:
Phosphorylation
Gene Symbol:
- SRC
- ASV
- c-src
- p60-Src
- SRC1
- c-SRC
- pp60c-src
Product Name:
phospho-Src (Tyr418) STAR ELISA Kit
Entrez Gene Number:
Materials Required but Not Delivered:
1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 µL
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 µL
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:
Src