Detection of phosphorylated signaling proteins using phospho-specific monoclonal antibodies (P1115)

Abstract

Abstract Triggering of extracellular surface receptors results in the initiation of numerous intracellular signaling cascades that are propagated via phosphorylation of specific tyrosine, serine, and/or threonine residues on signaling proteins. Until recently, analysis of intracellular phospho-signaling relied solely upon western blotting. The development of phospho-specific antibodies for use in flow cytometry has become a powerful tool for the detection of signaling events and has numerous advantages over western blotting; it is faster, requires fewer cells, and can be performed in a heterogeneous population of cells in conjunction with surface marker staining to allow further phenotyping of each population present in the sample. To this end, we have developed monoclonal antibodies specific for phosphorylation of various signaling molecules such as ERK1/2 (T202/Y204), STAT1 (Y701), STAT4 (Y693), and STAT5 (Y695). Here, we show the detection of phosphorylation of different intracellular signaling by flow cytometry across species in a treatment- and cell type-specific manner. One issue complicating phospho-specific flow cytometry is the inability to detect certain surface markers because of either epitope or fluorochrome destruction using the current, standard staining protocol. We show options that allow multi-parametric staining with different phospho-specific antibodies.