Receptor Tyrosine Kinases
The WideScreen RTK panel includes comprehensive RTK target coverage for the Luminex xMAP platform. RTKs are transmembrane cell-surface kinases activated upon binding extracellular ligands. They are of great interest due to their control of critical developmental and disease processes.
The Novagen WideScreen RTK Assay allows single or multiplex analysis of key RTKs including EGFR, IGF-1R, HGFR, PDGFRb, HER-2, VEGFR2, and Tie-2. The oncological roles of several of these receptors have been intensely scrutinized.
For example, EGFR overexpression or mutation has been correlated with numerous solid organ tumors including colorectal, pancreatic, lung, and non-small-cell lung cancers and glioblastoma. HER-2 overexpression (occurring in 25% of breast cancer patients) is associated with poor prognosis and disease recurrence, while VEGFR2 regulates angiogenesis pathways.
WideScreen RTK Assays consist of a series of phosphotyrosine (pTyr)-specific RTK assays and companion Total (phosphorylated plus unphosphorylated) assays. pTyr assays utilize specific capture antibodies and a broad-spectrum, pTyr-detection antibody. Total RTK assays, which include standards, allow relative comparison of pTyr and Total RTK in the sample for each receptor when Total and pTyr assays are conducted in parallel.
WideScreen RTK Assay Kits include an optimized buffer for detergent-mediated lysis, eliminating the need to alter lysate preparation for each antibody/analyte combination. Complete Kits include all seven premixed, antibody-conjugated capture beads, detection antibodies, total RTK standards, extraction reagents, and wash buffers. Single Bead Kits are available for researchers wishing to define their own multiplex assay combinations.
WideScreen RTK assay applications include protein-expression profiling, RNAi knock-down experiment confirmation, biomarker quantitation, inhibitor profiling, pathway analysis, and compound screening. Figure 1A shows an example of protein-expression profiling conducted with the WideScreen RTK Total Assay Complete Kit.
In this experiment, HUVEC and HT-29 cell-line extracts were analyzed for protein-expression levels of all seven RTKs in the panel. The results indicate marked differences in RTK protein expression between the two lines. HUVEC cells showed a high expression of Tie-2, as has been previously reported. HT-29 human colon cancer cells displayed a characteristically high level of HER-2 protein expression.
Figure 1B demonstrates the use of the WideScreen pTyr Assay to assess the response of serum-starved HT-29 cells to increasing concentrations of PD 168393, known to be highly specific for EGFR and HER-2.