Biology Reference
In-Depth Information
CHAPTER
8
Going Solo: Measuring the
Motions of Microtubules with
an In Vitro Assay for TIRF
Microscopy
Kris Leslie and Niels Galjart
Department of Cell Biology, Erasmus Medical Center, P.O. Box 2040, Rotterdam, The Netherlands
CHAPTER OUTLINE
Introduction ............................................................................................................ 110
8.1 Protocols......................................................................................................... 111
8.1.1 Preparation of Coverslips and Slides ................................................ 111
8.1.1.1 Chemical Cleaning of Slides and Coverslips .............................. 111
8.1.1.2 Sonication of Slides and Coverslips .......................................... 111
8.1.2 Assembly of in vitro Sample Chambers for the TIRF Microscope ......... 112
8.1.3 Preparation of GMPCCP-stabilized MT Seeds .................................... 113
8.1.4 TIRF Microscopy of þTIPs in vitro.................................................... 115
8.1.4.1 Preparation of an Oxygen Scavenger System ............................ 116
8.1.4.2 Chemically Functionalizing the Sample Chamber ...................... 117
8.1.4.3 Attaching the Seeds and Blocking the Chamber ....................... 118
8.1.4.4 Preparation of the Protein Samples .......................................... 120
8.1.4.5 TIRF Microscopy of the Samples.............................................. 121
Concluding Remarks ............................................................................................... 123
Acknowledgments ................................................................................................... 123
References ............................................................................................................. 123
Abstract
Microtubule (MT) plus-end-tracking proteins (
TIPs) specifically associate with the
plus ends of growingMTs, thereby determining, in many different ways, the dynamic
behavior of the MTs. Over the past years, a variety of fluorescently tagged
þ
TIPs
have been purified. Their reconstitution together with other purified components
þ
