Biology Reference
In-Depth Information
CONCLUDING REMARKS
The methods described here for the
in vitro
MT assay have been used for a number of
years at Erasmus MC. They are easily adaptable for use either in ensemble or single-
molecule experiments. In this chapter, we have tried to describe the protocols in
enough practical detail so as to make them simple to follow. For theoretical discus-
sions on the molecular mechanisms that govern the motions of MTs and
TIPs, we
refer to recent reviews (
Akhmanova & Steinmetz, 2010; Galjart, 2010
). The key to
successful
in vitro
MT experiments is adequate preparation of the reconstituted com-
ponents. One should spend time ensuring that the tubulins,
þ
TIPs, and other factors
that are used in the assay are purified to a high standard and are well characterized;
otherwise, the reproducibility of the system will degrade significantly and/or results
from the assay may be interpreted incorrectly.
þ
Acknowledgments
This work is financed by the Netherlands Organisation for Scientific Research (NWO,
CW-ECHO).
We would like to thank Dr Jeffrey Van Haren for kindly providing us with an image of EB1
comets from HeLa cells.
References
