Tubulin in vitro polymerization assay

CG Christoph Grohmann
FW Francesca Walker
MD Mark Devlin
ML Meng-Xiao Luo
AC Anderly C. Chüeh
JD Judy Doherty
FV François Vaillant
GH Gwo-Yaw Ho
MW Matthew J. Wakefield
CW Clare E. Weeden
AK Alvin Kamili
JM Jayne Murray
SP Sela T. Po’uha
JW Janet Weinstock
SK Serena R. Kane
MF Maree C. Faux
EB Esmee Broekhuizen
YZ Ye Zheng
KS Kristy Shield-Artin
NK Nadia J. Kershaw
CT Chin Wee Tan
HW Helen M. Witchard
GE Gregor Ebert
SC Susan A. Charman
IS Ian Street
MK Maria Kavallaris
MH Michelle Haber
JF Jamie I. Fletcher
MA Marie-Liesse Asselin-Labat
CS Clare L. Scott
JV Jane E. Visvader
GL Geoffrey J. Lindeman
KW Keith G. Watson
AB Antony W. Burgess
GL Guillaume Lessene
ask Ask a question
Favorite

A commercial fluorescence-based tubulin polymerization assay (Cytoskeleton, Denver, CO; Cat. # BK011P) was used as per manufacturer’s instructions for detection of tubulin polymerization inhibitors. Briefly, porcine tubulin (3 mg/ml tubulin in 80 mM PIPES pH 7.0, 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP and 10% glycerol) was incubated at 37 °C with either vehicle control (DMSO), with Paclitaxel or antimitotic compound. Each condition was tested in duplicate. Polymerization was measured over time by excitation at 360 nm and emission at 420 nm in a temperature controlled 96-well plate fluorimeter (Envision 2105, Perkin-Elmer, Waltham, MS) equipped with filters for excitation at 340–360 nm and emission at 420–450 nm.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A