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Thermostabilization, Expression, rening och kristallisation av den mänskliga Serotonin Transporter Bound till<em> S</em> -citalopram
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Capitoli
- 00:05Titolo
- 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
Detta manuskript beskriver hur man screena för thermostabilizing mutationer, rena mänskliga serotonintransportören, generera antikroppar med hög affinitet, och kristallisera serotonintransportören-antikroppskomplex bundet till det antidepressiva läkemedlet S -citalopram. Detta protokoll kan anpassas till studier av andra utmanande membrantransportörer, receptorer, och kanaler.
Tags
Serotonin TransporterS-citalopramThermostabilizationExpressionPurificationCrystallizationMembrane ProteinsDrug TargetsNeurotransmitter TransportersReceptorsChannelsSoluble ProteinsHEK293S GnTIDNA TransfectionCitalopramC12MCHSProtease InhibitorsTritiated CitalopramBSAHis-tag AffinitySPA BeadsSertraline
