Protein-Ligand Interactions pp 439-453

Part of the Methods in Molecular Biology book series (MIMB, volume 1008)

Quantitative Fluorescence Co-localization to Study Protein–Receptor Complexes

  • Shanica N. Pompey
  • Peter Michaely
  • Katherine Luby-Phelps
Protocol

Abstract

Fluorescence microscopy can be used to assess quantitatively the interaction between a ligand and its receptor, between two macromolecules, or between a macromolecule and a particular intracellular compartment by co-localization analysis. In general, this analysis involves tagging potential interacting partners with distinct fluorophores—by direct labeling of a small ligand, by expression of fluorescent cDNA constructs, by immunofluorescence labeling, or by some combination of these methods. Pairwise comparison of the fluorescence intensity of the two fluorophores at each pixel in a two channel digital image of the sample reveals regions where both are present. With appropriate protocols, the image data can be interpreted to indicate where the potential interacting partners are co-localized. Keeping in mind the limited resolution of the light microscope, co-localization is often used to support the claim that two molecules are interacting.

All quantitative methods for evaluating co-localization begin with identifying the pixels where the intensities of both color channels are above background. Typically this involves two sequential image segmentation steps: the first to exclude pixels where neither channel is above background, and the second to set overlap thresholds that exclude pixels where only one color channel is present. Following segmentation, various quantitative measures can be computed to describe the remaining subset of pixels where the two color channels overlap. These metrics range from simple calculation of the fraction of pixels where overlap occurs to more sophisticated image correlation metrics. Additional constraints may be employed to distinguish true co-localization from random overlap. Finally, an image map showing only the co-localized pixels may be displayed as an additional image channel in order to visualize the spatial distribution of co-localized pixels. Several commercial and open source software solutions provide this type of co-localization analysis, making image segmentation and calculation of metrics relatively straightforward. As an example, we provide a protocol for the time-dependent co-localization of fluorescently tagged lipoproteins with LDL receptor (LDLR) and with the early endosome marker EEA1.

Key words

Fluorescence Co-localization Confocal Deconvolution Immunofluorescence Digital imaging LDL VLDL LDL receptor 

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    Image documentation for JaCoP can be found here: http://imagejdocu.tudor.lu/lib/exe/fetch.php?media=plugin:analysis:jacop_2.0:just_another_colocalization_plugin:jacop_ijconf2008.pdf Documentation and a link to the source code for Coloc 2, Colocalization Threshold and Colocalization Test can be found here: http://fiji.sc/wiki/Colocalization_Analysis

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Shanica N. Pompey
    • 1
  • Peter Michaely
    • 1
  • Katherine Luby-Phelps
    • 1
  1. 1.Department of Cell BiologyUT Southwestern Medical SchoolDallasUSA

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