Biology Reference
In-Depth Information
CHAPTER
19
Methods for Studying
Microtubule Binding Site
Interactions: Zampanolide
as a Covalent Binding Agent
Jessica J. Field
*
, Enrique Calvo
{
, Peter T. Northcote
{
, John H. Miller
*
,
Karl-Heinz Altmann
}
and Jos ´ Fernando D´az
}
*
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
{
Unidad de Proteo´mica, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
{
School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington,
New Zealand
}
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences,
Swiss Federal Institute of Technology, Zu¨rich, Switzerland
}
Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
CHAPTER OUTLINE
Introduction ............................................................................................................ 304
19.1 Materials.....................................................................................................305
19.1.1 Tubulin and Stabilized Microtubules ....................................... 305
19.1.2 Buffers ................................................................................. 306
19.1.3 Software for Data Analysis...................................................... 307
19.1.4 Ultracentrifugation ................................................................ 307
19.2 Methods and Results....................................................................................307
19.2.1 Induction of Microtubule Assembly ......................................... 307
19.2.1.1 Microtubule Assembly in Glycerol Buffer .......................308
19.2.1.2 Microtubule Assembly in PEDTA Buffer.........................309
19.2.1.3 Transmission Electron Microscopy ................................310
19.2.2 Flutax Displacement Assay ..................................................... 310
19.2.3 Laulimalide/peloruside Competition Assay ............................... 312
19.2.3.1 Notes ...........................................................................314
19.2.4 Stoichiometry........................................................................ 314
19.2.5 Binding Kinetics.................................................................... 316
19.2.5.1 Flutax-2 Method ...........................................................316
19.2.5.2 High-performance Liquid Chromatography Analysis .......317
