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Chapters
- 00:05Title
- 02:28Fly Cup Preparation and Embryo Collection
- 04:00Embryo Homogenization and Clarification
- 04:48Microtubule Polymerization, Kinesin Binding and Differential Sedimentation of Microtubules and Kinesin
- 06:49Representative Kinesin Purification Results
- 08:29Conclusion
Dette er en protokol til at isolere aktive fuld længde kinesin fra<em> Drosophila</em> Embryoner til enkelt-molekyle biofysiske undersøgelser. Vi viser, hvordan at indsamle embryoner, gør embryo lysatet, og derefter polymerisere mikrotubuli (MT). Kinesin oprenses ved at immobilisere det på MT-, spinde ned kinesin-MT komplekser, og derefter at frigive kinesin fra MT via ATP tilsætning.
Tags
IsolationPurificationKinesinDrosophila EmbryosMotor ProteinsMicrotubulesSub-cellular LocationsNeurodegenerative DiseasesTherapeutic ApproachesATP HydrolysisPurification ProtocolGeneticsSingle-molecule Biophysical StudiesLaying CupsFemales Per CupOvernight CollectionsBleach DechorionatedHomogenizedLow Speed SpinHigh Speed CentrifugationClarified SupernatantGTP And TaxolPolymerize MTsImmobilized On Polymerized MTsATP AnalogAdenylyl ImidodiphosphateRoom TemperatureMicrotubule Pellet
