EGFR pTyr Bead Kit
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19 June 2013
This product has been discontinued.
Bead-based flow cytometric assays enable sensitive, precise quantification of analytes within a sample. When directed towards protein analytes, such assays are essentially ELISAs on a bead. Samples are combined with labeled microparticles covalently conjugated to a capture antibody. Analytes captured on the beads are identified with detection antibodies and a fluorescent label. A major advantage over traditional protein analyte quantification methods (such as ELISA) is the capacity for multiplexing, as bead-based assays allow simultaneous quantification of multiple analytes in a small sample volume.
Receptor tyrosine kinases (RTKs) are critical regulators of numerous cell signaling pathways and have been implicated in various disease states. Ligand binding to the extracellular domain of transmembrane RTKs triggers receptor dimerization and autophosphorylation of an intracellular kinase domain. This event ultimately triggers activation of downstream pathway proteins via phosphotyrosine-SH2 domain interactions. The WideScreen® RTK Assay Kits allow for the simultaneous quantification of any of the following RTKs:
• Epidermal Growth Factor Receptor (EGFR)
• Insulin-like Growth Factor 1 Receptor (IGF-1R)
• Hepatocyte Growth Factor Receptor (HGFR)
• Platelet-Derived Growth Factor Receptor beta (PDGFRβ)
• Human Epidermal Growth Factor Receptor 2 (HER-2)
• Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)
• Tyrosine Kinase with Immunoglobulin and EGF Repeats 2 (Tie-2)
The WideScreen® RTK Assay workflow is shown in the figure at the bottom of this page. If expressed in the cell line of interest, individual RTK proteins may be present in either inactive or active states depending on availability of the RTK ligand. Cellular protein samples are prepared by gentle membrane extraction. Extracts are combined with dye-labeled Luminex® xMAP® beads covalently conjugated to capture antibodies specific to each RTK. After incubation and several washes, the beads are incubated with biotin-labeled detection antibodies. The resulting bead-immobilized immunosandwich is detected with streptavidin-phycoerythrin and quantified using a Luminex® xMAP® instrument.
Importantly, detection antibodies differ between the WideScreen® Total RTK assay and the WideScreen® pTyr RTK assay. In the Total assay, the detection antibody recognizes a second epitope on the RTK, allowing quantification of RTK levels without regard to phosphorylation state. In the pTyr assay, the detection antibody is specific to conserved pTyr on all RTKs. Relative quantification of RTK phosphorylation (expressed in median fluorescence intensity units) is possible using the pTyr kit. Because antibodies would compete for the same analyte, the RTK Total Assays cannot be multiplexed with the RTK pTyr assays.
Applications of the WideScreen® RTK Assay Kits include:
• Biomarker quantification
• Expression profiling
• Confirmation of knock-down experiments
• RTK agonist or antagonist profiling
• Pathway analysis
• High-throughput compound library screening
For maximum flexibility and user-defined multiplex assay configuration, components of the WideScreen® RTK 1 Total Assay Complete Kit and the WideScreen® RTK 1 pTyr Assay Complete Kit are available separately. All components are necessary for carrying out the RTK bead-based assays. The RTK Bead Kits and buffers are not compatible with other bead kits and reagents sold by Novagen or other vendors.
The WideScreen® RTK 1 Total Assay Complete Kit includes the entire set of reagents to run 96 test wells, including the RTK Total 7-plex, RTK Total Standards Mix, Cell Extraction Kit, and WideScreen® Reagent Kit.
The WideScreen® RTK 1 pTyr Assay Complete Kit comprises the entire set of reagents to run 96 test wells, including the RTK pTyr 7-plex, Cell Extraction Kit, and WideScreen® Reagent Kit. No standards are included in the pTyr Assay Complete Kit.
RTK Bead Kits contain antibody-coated Capture Beads and biotinylated Detection Antibodies used for target detection via immunoassay sandwiches. Bead Kits may be purchased separately, or as pre-mixed panels. Available Bead Kits are listed in Appendix A of Technical Bulletin (TB) 500. Performance specifications for the Bead Kits are detailed in the individual Certificates of Analysis, available online. Each RTK Bead Kit contains sufficient reagents for 100 tests.
The RTK recombinant standards are used to create standard curves when performing quantitative assays. Each individual RTK Total Bead Kit (used to quantify a specific RTK target regardless of phosphorylation state) includes the appropriate individual recombinant standard. Pre-mixed RTK recombinant standards are supplied with the RTK Total Assay Complete Kit and the RTK Total 7-Plex. The concentration of standards in the standard curves can be found in Appendix B of TB500. Each recombinant standards mix or individual standard contains reagents sufficient to generate eight singleplex or multiplex standard curves, or four standard curves in duplicate.
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