Biology Reference
In-Depth Information
CHAPTER
1
Kamalika Mukherjee and Susan L. Bane
Department of Chemistry, Binghamton University, State University of New York, Binghamton,
New York, USA
Site-Specific Fluorescent
Labeling of Tubulin
CHAPTER OUTLINE
Introduction ................................................................................................................ 2
1.1 Material .............................................................................................................. 4
1.1.1 TLL Expression and Purification .......................................................... 4
1.1.2 Labeling Tubulin ................................................................................ 4
1.1.3 Detection of Labeled Tubulin............................................................... 5
1.1.4 Detection of Polymerization Activity ..................................................... 5
1.2 Method................................................................................................................ 5
1.2.1 Protein Isolation and Purification......................................................... 5
1.2.1.1 Tubulin.........................................................................................5
1.2.1.2 Tubulin Tyrosine Ligase ................................................................6
1.2.2 Tubulin Detyrosination ........................................................................ 6
1.2.3 Coupling Tyrosine Derivative to the C-terminus of a-tubulin.................... 6
1.2.3.1 Ligand Preparation .......................................................................6
1.2.3.2 Coupling Reaction ........................................................................7
1.2.4 Fluorescent Labeling of Tubulin........................................................... 7
1.2.4.1 Preparation of Catalyst Stock.........................................................7
1.2.4.2 Preparation of Fluorophore Stock ..................................................7
1.2.4.3 Preparation of fluorophore-labeled Tubulin ....................................8
1.2.5 Coupling Biotin to the C-terminus of a-tubulin ...................................... 8
1.2.5.1 BH Stock Preparation ...................................................................8
1.2.5.2 Biotinylation of Tubulin .................................................................8
1.2.6 Detection of Modification in the C-terminus of Tubulin .......................... 8
1.2.6.1 Detection of Tyrosine Or 3-fomyltyrosine in the C-terminus
of
a
-tubulin ...............................................................................................8
1.2.6.2 Detection of fluorophore-labeled Tubulin .......................................9
1.2.6.3 Detection of Biotinylated Tubulin...................................................9
1.2.7 Evaluation of Tubulin Assembly Activity ............................................... 9
Acknowledgments ..................................................................................................... 11
References ............................................................................................................... 11
