Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

J Vis Exp. 2017 Aug 10:(126):55996. doi: 10.3791/55996.

Abstract

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Cell Line
  • Fluorescence
  • Fluorescent Antibody Technique / methods
  • Fluorescent Dyes / chemistry
  • Golgi Apparatus / chemistry*
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism
  • HeLa Cells
  • Humans
  • Microscopy, Confocal / instrumentation
  • Microscopy, Confocal / methods*
  • Molecular Imaging / methods*
  • Nocodazole / pharmacology
  • Proteins / analysis*
  • Proteins / chemistry
  • Signal-To-Noise Ratio

Substances

  • Fluorescent Dyes
  • Proteins
  • Nocodazole