71943  WideScreen^® RTK pTyr Assay Complete Kit

Bead-based flow cytometric xMAP^® assays enable sensitive, precise detection of analytes within a sample. When directed toward proteins, such assays are essentially sandwich immunoassays on a bead. Samples are combined with fluorescently labeled microparticles (beads) covalently conjugated to a capture antibody. Analytes captured on the beads are identified with detection antibodies and a fluorescent label. A major hallmark of bead-based assays over traditional protein detection methods (such as ELISA) is the capacity for multiplexing, as bead-based assays allow simultaneous detection of multiple analytes in a small sample volume. Other advantages of xMAP^® assays include flexibility, robustness, simple sample handling, and requirement of minimal sample volumes, making them an ideal platform for immunodetection of biomarkers and signaling proteins.

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 provides users with the ability to quantify the following RTKs, in either its phosphorylated or total state:
• Epidermal Growth Factor Receptor (EGFR)
• Hepatocyte Growth Factor Receptor (HGFR)
• Human Epidermal Growth Factor Receptor 2 (HER-2)
• Insulin-Like Growth Factor 1 Receptor (IGF-1R)
• Platelet-Derived Growth Factor Receptor beta (PDGFRβ)
• Tyosine Kinase with Immunoglobulin and EGF Repeats 2 (Tie-2)
• Total Vascular Endothelial Growth Factor Receptor 2 )

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. In the WideScreen^® assay, cellular protein samples are prepared by using a Cell Extraction Kit and WideScreen^® Reagent Kit, both of which can be purchased separately or are already included in the WideScreen^® RTK Total Assay Complete Kit and the WideScreen^® RTK pTyr Assay Complete Kit. Extracts are combined with dye-labeled Luminex^® xMAP^® beads covalently conjugated to capture antibodies specific to each phosphorylation site. 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 acquired 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 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. The WideScreen^® Total Assay Complete Kit and the WideScreen^® RTK pTyr Assay Complete Kit comprises the entire set of reagents to run 96 test wells for EGFR, HGFR, HER-2, IGF-1R, PDGFRβ, Tie-2, and VEGFR2, including the RTK pTyr 7-plex in either its phosphorylated or total state, a Cell Extraction Kit, and a WideScreen^® Reagent Kit. Standards are included in the WideScreen^® Total Assay Complete Kit, but not the WideScreen^® pTyr Assay Complete Kit The RTK recombinant standards are used to create standard curves when performing quantitative assays. For a table of standard concentrations, please refer to TB500 read the appendices carefully. Each recombinant standards mix contains reagents sufficient to generate eight singleplex or multiplex standard curves, or four standard curves in duplicate.