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Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity

  • Laura Ren Huey Ip
  • Christina Anja GewinnerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

Abstract

Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.

Key words

Phosphatidylinositol Inositol-polyphosphate-4-phosphatase type II PTEN PI-3 kinase Lipid phosphatase assay Cell signaling Phosphorylation Enzyme assay Thin-layer chromatography Phosphoinositide GST-pull-down assay Insect cell culture Mammalian overexpression system 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Research Department of Cancer Biology, UCL Cancer Institute, Paul O’Gorman BuildingUniversity College LondonLondonUK
  2. 2.Translational Innovation Group, UCL-Eisai CollaborativeUniversity College LondonLondonUK

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