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热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em>取值</em> -citalopram
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram
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热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em>取值</em> -citalopram

DOI:
10.3791/54792-v

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12:21 min

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November 27, 2016

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, ,
1Vollum Institute,Oregon Health & Science University, 2Graduate Group in Biophysics,University of California, San Francisco, 3Howard Hughes Medical Institute,Oregon Health & Science University

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

Summary

Automatic Translation

Automatic Translation

这份手稿介绍如何筛选thermostabilizing突变，净化人的羟色胺转运，产生高亲和力的抗体，并结晶势必抗抑郁药物小号 -citalopram羟色胺转运-抗体复合物。该协议可以适于其他具有挑战性的膜转运，受体和通道的研究。

Tags

Serotonin TransporterS-citalopramThermostabilizationExpressionPurificationCrystallizationMembrane ProteinsDrug TargetsNeurotransmitter TransportersReceptorsChannelsSoluble ProteinsHEK293S GnTIDNA TransfectionCitalopramC12MCHSProtease InhibitorsTritiated CitalopramBSAHis-tag AffinitySPA BeadsSertraline

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