The Pathscan® Multiplex Western Detection Kit offers a unique method to assay the phosphorylation of multiple proteins on one membrane without stripping and reprobing. This method saves the user valuable time while increasing accuracy and minimizing reagent waste. The system allows the user to simultaneously detect the phosphorylation of PDGF Receptor β, SHP2, Akt, and p44/42 MAPK proteins in response to PDGF. The kit also includes an eIF4E antibody to control protein loading. In addition, each Pathscan® Multiplex Western Detection Kit contains treated and untreated cell lysates and the Phototope®-HRP Western Detection System. The kit includes enough primary and secondary antibodies to perform five Western blot experiments.
Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The eIF4E control antibody detects total levels of eIF4E independent of phosphorylation state to determine protein loading. All of the antibodies detect endogenous levels of the target proteins.
Antibodies are produced by immunizing rabbits with synthetic peptides, and are purified by combinations of Protein A and peptide affinity chromatography.
Platelet-derived growth factor (PDGF) is a dimeric molecule that exists as homodimers or heterodimers of related polypeptide chains (A and B). Two types of PDGF receptors have been identified. The PDGF alpha-receptor binds all three isoforms with high affinity, whereas the beta-receptor binds only PDGF-BB with high affinity, PDGF-AB with low affinity and does not appear to bind PDGF-AA (1). PDGF exerts its stimulatory effects on cells by binding to these two related protein tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation, allowing binding and activation of cytoplasmic SH2-domain-containing signal transduction molecules. Thereby, a number of different signaling pathways are initiated, leading to cell growth, actin reorganization, migration and differentiation (2-4). In clinical studies, PDGF expression has been shown in a number of different solid tumors, from glioblastomas to prostate carcinomas. In these various tumor types, the biologic role of PDGF signaling can vary from autocrine stimulation of cancer cell growth to more subtle paracrine interactions involving adjacent stroma and even angiogenesis. Targeting PDGF signaling becomes an effective way for tumor treatment (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc. LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
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