Biology Reference
In-Depth Information
CHAPTER
12
Witchuda Saengsawang
*
and Mark M. Rasenick
{,
{
Heterotrimeric G Proteins
and Microtubules
*
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
{
Departments of Physiology & Biophysics and Psychiatry, University of Illinois at Chicago,
Chicago, Illinois, USA
{
The Jesse Brown VA Medical Center, Chicago, Illinois, USA
CHAPTER OUTLINE
Introduction ............................................................................................................ 174
12.1 General Protocols ...........................................................................................174
12.1.1 In Vitro Determination of the Association Between Tubulin and Gsa 174
12.1.1.1 Protein Purification ............................................................ 175
12.1.1.2 Pull-down Assays............................................................... 177
12.1.1.3 Binding Analysis By SPR ................................................... 177
12.1.1.4 Peptide Array Membrane Analysis...................................... 178
12.1.2 Functional Consequence of Tubulin-Gsa Interaction...................... 179
12.1.2.1 Steady-state Tubulin GTPase Assay.................................... 179
12.1.2.2 Single-turnover Tubulin GTPase Assay ............................... 180
12.1.3 Functional Consequences of Gsa-mediated Activation of Tubulin
GTPase: Gsa and Gsa-derived Peptides Increase Microtubule
Dynamics ............................................................................. 181
12.1.3.1 Microtubule Polymerization Assay ...................................... 181
12.1.3.2 Microtubule Dynamics Assay By Video Microscopy............. 181
12.1.4 Cellular Consequences of Gsa Interaction With
Tubulin/microtubules ............................................................ 183
12.1.4.1 Binding Studies in Cells ..................................................... 183
12.1.4.2 Microtubule Dynamics Assay in Cells ................................. 185
12.1.4.3 Cell Morphology Assay ....................................................... 187
12.2 Buffer Compositions ........................................................................................187
Concluding Comments ............................................................................................. 188
Acknowledgments ................................................................................................... 188
References ............................................................................................................. 188
