Biology Reference
In-Depth Information
CHAPTER
13
Purification and
Biophysical Analysis
of Microtubule-Severing
Enzymes In Vitro
Juan Daniel Diaz-Valencia
*
, Megan Bailey
{
and Jennifer L. Ross
*
*
Department of Physics, University of Massachusetts Amherst, Amherst, Massachusetts, USA
{
Molecular and Cellular Graduate Program, University of Massachusetts Amherst,
Amherst, Massachusetts, USA
CHAPTER OUTLINE
Introduction ............................................................................................................ 192
Importance of in Vitro Biochemical and Biophysical Studies .................................... 194
13.1 Methods .........................................................................................................194
13.1.1 Purification From Sf9 Cells ......................................................... 195
13.1.1.1 Amplifying Virus................................................................. 195
13.1.1.2 Protein Expression ............................................................. 196
13.1.1.3 Protein Purification ............................................................ 196
13.1.1.4 Concentration Determination.............................................. 198
13.1.1.5 Special Considerations for Severing Enzymes ..................... 198
13.1.2 Required Elements for Biophysical Assays .................................... 200
13.1.2.1 Microtubules ..................................................................... 200
13.1.2.2 Experimental Chamber....................................................... 202
13.1.3 Biophysical Assays ..................................................................... 203
13.1.3.1 Epifluorescence and Single Molecule TIRF Imaging
methods .......................................................................................... 204
13.1.3.2 Two-color Colocalization..................................................... 205
13.1.3.3 Severing and Depolymerization .......................................... 207
13.1.3.4 Binding and Diffusion ........................................................ 207
13.1.3.5 Photobleaching.................................................................. 208
Discussion and Summary......................................................................................... 211
Acknowledgments ................................................................................................... 211
References ............................................................................................................. 211
