Biology Reference
In-Depth Information
CHAPTER
11
Tubulin-Specific
Chaperones: Components
of a Molecular Machine
That Assembles the a / b
Heterodimer
Guoling Tian and Nicholas J. Cowan
Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical
Center, New York, New York, USA
CHAPTER OUTLINE
Introduction ............................................................................................................ 156
11.1 Methods .........................................................................................................159
11.1.1 cDNA Clones and Vectors ............................................................ 159
11.1.2 Expression of TBCA, TBCB, and TBCC ......................................... 161
11.1.2.1 Bacterial Cell Growth and Induction ................................... 161
11.1.2.2 Preparation of Bacterial Lysates ......................................... 162
11.1.3 Purification of TBCA, TBCB, and TBCC ........................................ 162
11.1.3.1 Chromatography on Either Sepharose Q (TBCA and TBCB)
or Sepharose S High-Performance (TBCC) Ion Exchange Resins ....... 162
11.1.3.2 Anion Exchange Chromatography on MonoQ...................... 163
11.1.3.3 Gel Filtration Chromatography (optional)............................. 163
11.1.4 Expression of TBCE .................................................................... 163
11.1.4.1 Construction of Recombinant Baculovirus .......................... 163
11.1.5 Purification of TBCE................................................................... 164
11.1.5.1 Preparation of Insect Cell Lysates....................................... 164
11.1.5.2 Anion Exchange Chromatography....................................... 164
11.1.5.3 Chromatography on Hydroxylapatite ................................... 165
11.1.5.4 Size Exclusion Chromatography (optional) .......................... 165
11.1.6 Expression of TBCD .................................................................... 165
11.1.7 Purification of TBCD................................................................... 166
11.1.7.1 Preparation of HeLa Cell Lysates ........................................ 166
11.1.7.2 Anion Exchange Chromatography on Q Sepharose High
Performance .................................................................................... 166
11.1.7.3 Anion Exchange Chromatography on MonoQ...................... 166
 
Search WWH ::




Custom Search