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Chapters
- 00:05Title
- 00:47Transfection of Adherent HEK293S GnTI-cells for Thermostability Screen
- 02:24Scintillation Proximity-based Thermostability Screen with Citalopram
- 04:18Expression of the Human Serotonin Transporter in HEK293S GnTI-cells
- 05:24Affinity-purification of the Serotonin Transporter for Immunization and Crystallization
- 07:12Formation of Transporter-antibody Complexes and Separation by Size-exclusion Chromatography
- 08:21Crystallization of Transporter-antibody Complexes by Hanging Drop
- 10:01Results: Thermostabilization and Crystallization of the Serotonin Transporter in an Antidepressant-bound State
- 11:25Conclusion
Dit manuscript beschrijft hoe om te screenen op thermostabilizing mutaties, zuiveren de menselijke serotonine transporter, het genereren van antilichamen met hoge affiniteit, en kristalliseren de serotonine transporter antilichaam-complex gebonden aan het antidepressivum citalopram S. Dit protocol kan worden aangepast aan de studie van andere uitdagende membraantransporteiwitten, receptoren en kanalen.
Tags
Serotonin TransporterS-citalopramThermostabilizationExpressionPurificationCrystallizationMembrane ProteinsDrug TargetsNeurotransmitter TransportersReceptorsChannelsSoluble ProteinsHEK293S GnTIDNA TransfectionCitalopramC12MCHSProtease InhibitorsTritiated CitalopramBSAHis-tag AffinitySPA BeadsSertraline
