Biology Reference
In-Depth Information
CHAPTER
14
Measurement of In Vitro
Microtubule Polymerization
by Turbidity and
Fluorescence
Matthew Mirigian
*
, Kamalika Mukherjee
{
, Susan L. Bane
{
and Dan L. Sackett
*
*
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health
and Human Development, NIH, Bethesda, Maryland, USA
{
Department of Chemistry, Binghamton University, State University of New York, Binghamton,
New York, USA
CHAPTER OUTLINE
14.1 Background and Theory.................................................................................. 216
14.1.1 Background .............................................................................. 216
14.1.2 Critical Concentration ................................................................ 217
14.1.3 Polymerization Curves and the “Turbidity Coefficient” .................. 219
14.1.4 Polymerization Promoters........................................................... 222
14.1.5 Measurement of Drug Effects—Polymerization Inhibitors .............. 223
14.2 Materials and Equipment ................................................................................ 223
14.2.1 Equipment................................................................................ 224
14.2.2 Materials .................................................................................. 225
14.3 Methods ........................................................................................................ 225
14.3.1 Preparation of Tubulin ............................................................... 225
14.3.2 Turbidity Assay in Cuvettes ........................................................ 225
14.3.3 Turbidity Assay in Multiwell Plates.............................................. 226
14.3.4 Analysis of Turbidity .................................................................. 227
14.3.5 Fluorescence Assay in Multiwell Plates........................................ 227
References ............................................................................................................. 228
Abstract
Tubulin polymerization may be conveniently monitored by the increase in turbidity
(optical density, or OD) or by the increase in fluorescence intensity of diamidino-
phenylindole. The resulting data can be a quantitative measure of microtubule
(MT) assembly, but some care is needed in interpretation, especially of OD data.
Methods in Cell Biology, Volume 115
