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Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software
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Bioquímica
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JoVE Journal Bioquímica
Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

DOI:
10.3791/58157-v

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08:22 min

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July 17, 2018

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, , , , ,
1Cellular Imaging Group, Wellcome Centre Human Genetics,University of Oxford, 2Division of Structural Biology, Wellcome Centre Human Genetics,University of Oxford, 3Dynamic Structural Virology Group,Biocruces Health Research Centre, 4IKERBASQUE,Basque Foundation for Science

Capítulos

  • 00:04Título
  • 00:50FKBP12F36V-mVenus Purification
  • 03:24Preparation of Multiwell Plate Array
  • 04:19Calibration-free Confocal Acquisition
  • 06:26Results: FKBP12 Dimerizes upon Addition of BB Dimerizer Drug
  • 07:23Conclusion

Summary

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This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Tags

Keywords: Protein Homo-oligomerizationIn Vitro QuantificationCalibration-freeConfocal MicroscopyBrightness AnalysisProtein-protein InteractionsDrug ScreeningCancer DrugsSmall-molecule InhibitorsFusion InhibitorsProtein DynamicsCell-cell InteractionsFKBP12His6 TagMVenus TagIMACSize-exclusion Chromatography

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