The Signal Transduction Core Facility provides state-of-the-art expertise and instrumentation to research projects at OMRF. A number of specific techniques are described below.
Oklahoma Medical Research Foundation
Signal Transduction Core Facility
825 NE 13th Street
Oklahoma City, OK 73104
Phone: (405) 271-7209
Measurements of intracellular calcium
Changes in cytoplasmic calcium are a common feature of many signaling pathways, including those initiated by protein tyrosine kinases and by heterotrimeric GTP-binding proteins. The core lab is prepared for such measurements using Indo-1 fluorescence assessed by a Perkin-Elmer LS-50 spectrofluorimeter.
Measurements of protein interactions
Protein associations mediated by numerous interaction domains are a fundamental and defining feature in signal transduction induced by receptors of hematopoietic cells. Accordingly, a number of techniques have evolved to assess the precise nature and identity of interacting proteins in various signaling situations. The signal transduction core facility has extensive experience in these techniques, some of which are listed below with documentation of their use. Invariably, however, immunoblotting of interacting proteins is the method of choice for their detection. The signal transduction core facility housed shared PAGE and electrophoretic transfer equipment, all materials for immunoblotting, and a Licor Odyssey Infared Imaging System for immuno-based detection and quantitation of proteins and nucleic acids on filters. Protein domain pull-downs, Peptide and phosphopeptide pull-downs, Co-immunoprecipitation, Far-western analysis, small GTPases like Rac, CDC42, Rho family members.
Activation of tyrosine protein kinases is the most proximal event in signal transduction by immunoreceptors or growth factor receptors, and frequently leads to stimulation of other essential serine/threonine kinases. Thus, the induced catalytic activity of these kinases is often used as an indicator of receptor-triggered signal transduction. Many of these limiting features have been resolved for numerous Ser/Thr and Tyr kinases, and the personnel in the core facility have considerable experience in determining these variables when novel kinases are identified. Ser/Thr kinases – MAP kinases, Protein kinase C, Akt, Tyr kinase – family members of Src; Syk; JAK; Lipid kinase – PtdIns 3-kinase.
Transient and stable transfection of hematopoietic cells is a rapid and informative method of determining the relationship between proteins in a signaling pathway. Transfections of lymphocytes with a limiting amount of a reporter enzyme and co-transfection with a relatively large amount of a dominant-positive or -negative signaling enzyme mutant has been a successful strategy to explore a potential relationship between two enzymes. The core facility maintains several cultured lymphocytic and myelocytic cell line models, and has developed and optimized techniques for their transfection. All of this information and technology is available to OMRF investigators.