Library Science

A collection of cutting-edge methods for investigating G protein signaling from a variety of perspectives ranging from in vitro biochemistry to whole animal studies. Among the readily reproducible techniques presented are those for the purification of G proteins and effectors enzymes, assays of these purified G proteins and effector enzymes, and for the study of G proteins interactions with effectors in intact cells. Additional methods are provided for assaying G protein coupled receptor structure, function, and localization; for studying the physiological roles for endogenous G proteins; and for examining lipid and phosphate modifications of RGS proteins.

Authors: Smrcka, Alan V.

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Table of Contents

• Purification of Recombinant G Protein a Subunits from Escherichia coli (pp. 3-20)
  Greentree, Wendy K.; Linder, Maurine E.
• Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged a and ßy Subunits (pp. 21-38)
  Kozasa, Tohru
• Expression and Purification of Soluble Adenylyl Cyclase from Escherichia coli (pp. 39-54)
  Beeler, Jeff A.; Tang, Wei-Jen
• Purification of Phospholipase Cß and Phospholipase Ce from Sf9 Cells (pp. 55-64)
  Ghosh, Mousumi; Wang, Huan; Kelley, Grant G.; Smrcka, Alan V.
• Assay for G Protein-Dependent Activation of Phospholipase Cß Using Purified Protein Components (pp. 67-76)
  Ghosh, Mousumi; Smrcka, Alan V.
• Assays of Recombinant Adenylyl Cyclases Expressed in Sf9 Cells (pp. 77-86)
  Taussig, Ronald
• Measurement of G Protein-Coupled Receptor-Stimulated Phospholipase D Activity in Intact Cells (pp. 89-98)
  Walker, Stephanie J.; Brown, H. Alex
• Analysis of G Protein-Mediated Activation of Phospholipase C in Cultured Cells (pp. 99-102)
  Liu, Bo; Wu, Dianqing
• Intensive Mutational Analysis of G Protein-Coupled Receptors in Yeast (pp. 105-120)
  Celic, Andjelka; Connelly, Sara M.; Martin, Negin P.; Dumont, Mark E.
• Green Fluorescent Protein-Tagged ß-Arrestin Translocation as a Measure of G Protein-Coupled Receptor Activation (pp. 121-126)
  Ferguson, Stephen S. G.; Caron, Marc G.
• Detection of G Protein-Coupled Receptors by Immunofluorescence Microscopy (pp. 127-134)
  Hinkle, Patricia M.; Puskas, John A.
• Assay of G Protein-Coupled Receptor Activation of G Proteins in Native Cell Membranes Using [35S]GTPyS Binding (pp. 135-144)
  Bidlack, Jean M.; Parkhill, Amy L.
• Analysis of the Coupling of G12/13 to G Protein-Coupled Receptors Using a Luciferase Reporter Assay (pp. 145-150)
  Liu, Bo; Wu, Dianqing
• Selective Inhibition of G Protein-Mediated Pathways Using RGS Domains (pp. 153-168)
  Kozasa, Tohru; Ye, Richard D.
1. Ribozymes as Tools for Suppression of G Protein y Subunits (pp. 169-180)
   Robishaw, Janet D.; Guo, Zheng-ping; Wang, Qin
2. In Vivo Adenoviral-Mediated Gene Transfer of the ßARKct to Study the Role of Gßy in Arterial Restenosis (pp. 181-192)
   Iaccarino, Guido; Koch, Walter J.
3. Analysis of RGS Protein Palmitoylation (pp. 195-204)
   Bernstein, Leah S.; Linder, Maurine E.; Hepler, John R.
4. Methods for Measuring RGS Protein Phosphorylation by G Protein-Regulated Kinases (pp. 205-220)
   Hollinger, Susanne; Hepler, John R.
5. The Use of Green Fluorescent Proteins to View Association Between Phospholipase Cß and G Protein Subunits in Cells (pp. 223-232)
   Scarlata, Suzanne; Dowal, Louisa
6. Cellular Localization of GFP-Tagged a Subunits (pp. 233-246)
   Hynes, Thomas R.; Hughes, Thomas E.; Berlot, Catherine H.

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