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How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells
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Biochemistry
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JoVE Journal Biochemistry
How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

DOI:
10.3791/58588-v

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11:03 min

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January 07, 2019

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,
1Gynecologic Oncology Division,Stanford University School of Medicine

Chapters

  • 00:04Title
  • 01:20Cell Culture and Transfection
  • 02:31Imaging and Photoactivation
  • 05:45Imaging Analysis and Algorithm for Ratiometric Intensity-based Quantification of Photoactivation Efficiency
  • 09:09Results: Maximizing Photoactivation Efficiency
  • 09:50Conclusion

Summary

Automatic Translation

Automatic Translation

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

Tags

Photo-activatable Fluorescent ProteinsPA-GFPPA-CherryFluorescence MicroscopyProtein ExpressionQuantificationLive CellsInternal RulersGenetically Coupled Fluorescent Proteins

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