Biology Reference
In-Depth Information
10.3.2 Microtubules Elongated from Taxol Seeds Immobilized on
Kinesin-Coated Glass .................................................................. 148
10.3.2.1 Preparation of MAP-free Tubulin ........................................ 148
10.3.2.2 Fluorescent-Tubulin Labeling............................................. 148
10.3.2.3 Recombinant KHC Reconstitution ...................................... 148
10.3.2.4 Taxol-Stabilized Microtubule Seeds Polymerization ............. 149
10.3.2.5 Preparation of the Incubation Chambers ............................ 149
10.3.2.6 Binding of Microtubule Seeds and Microtubule
Elongation ........................................................................................ 149
10.4 Discussion and Future Prospects......................................................................150
Acknowledgments ................................................................................................... 151
References ............................................................................................................. 152
Abstract
Microtubules display a very dynamic behavior, and the presence of the guanosine-
triphosphate (GTP) cap at the plus ends of microtubules is essential to regulate mi-
crotubule dynamics.
Dimitrov et al. (2008)
showed that GTP-tubulin is present not
only at the plus ends but also in discrete locations along the microtubule lattice.
These GTP islands were proposed to contribute to rescue events. Studying the local-
ization of GTP-tubulin in microtubules is essential to better comprehend some core
aspects in the regulation of microtubule dynamics. In this chapter, we recapitulate
essential tools to study the GTP-tubulin using the recombinant antibody MB11 from
permeabilized cells to
in vitro
assays.
Reagents
AMPPNP (Sigma, Ref: A2647)
Alexa 488-labeled goat antihuman antibody (Jackson Laboratories,
Ref: 709-165-149)
Anti-tubulin antibody (DM1A; Sigma, Ref: T6199)
Anti-GTP-tubulin antibody (MB11)
ATP (Sigma, Ref: A3377)
Attofluor chamber (Invitrogen)
b
-Mercaptoethanol (Sigma, Ref: M6250)
Casein (Sigma, Ref: C6905)
Catalase (Sigma, Ref: C40)
Dithiothreitol (DTT; Sigma, Ref: D9163)
D
-Glucose (Sigma, Ref: D-3179)
DMSO (Sigma, Ref: 276855)
EGTA (EuroMedex, Ref: 1310-B)
Ethanol (Carlo Erba, Ref: 528151)
Gelatin (Sigma, Ref: G1393)
