71932 MilliporeHER-2 Total Bead Kit

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:

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: