Liz Greene and Steve Henikoff Lab Protocols

Construction of Plasmids

• Mutants, Chimeras and Truncated Motors.

Expression and Purification of Kinesin Motors

• Bacterial Expression of Motors

• Purification of Ncd Motor Domain Protein

• Purification of Kinesin-1 Motor Domain Protein

• Purification of Kar3 Motor Domain Protein

ATPase Assays

• Steady-State ATPase Assays

• Nucleotide Binding/Hydrolysis Assays

• Assays with ³²P-ATP

Microtubule Binding Assays

• Pelleting Assays

Motility Assays

• Motility using VE-DIC Microscopy

• Motility with Fluorescent MTs

• Motor Polarity using Fluorescent MTs

• Motor Polarity with Axoneme-MTs

Reagents

• Tubulin Preparation

• Microtubule Assembly

• Axoneme Preparation

• Axoneme-microtubule Preparation

Crystallization

• Crystallization Trials

• Motor Crystalization Conditions