Determination of absolute protein numbers in single synapses by a GFP-based calibration technique

Nat Methods. 2005 Sep;2(9):677-84. doi: 10.1038/nmeth783.


To build a quantitative model of molecular organization of neurons, it is essential to have information about the number of protein molecules at individual synapses. Here we developed a method to estimate absolute numbers of individual proteins at actual excitatory synapses by calibrating the fluorescence intensity of microspheres with single EGFP molecules. In cultured hippocampal neurons, we observed a monotonous increase of postsynaptic protein numbers per single synapse during neuronal differentiation and subsequent stabilization. At maturity we calculated that a single excitatory postsynaptic site contains 100-450 of individual postsynaptic proteins, such as PSD-95, GKAP, Shank and Homer. This narrow range of postsynaptic protein content suggests relatively simple stoichiometry of postsynaptic molecular organization. The EGFP-based calibration technique provides an unprecedented general method for estimating the amounts of proteins in macromolecular complexes.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Gene Expression Profiling / methods*
  • Green Fluorescent Proteins*
  • Hippocampus / metabolism
  • Mice
  • Microscopy, Fluorescence / methods*
  • Nerve Tissue Proteins / metabolism*
  • Neurons / metabolism*
  • Synapses / metabolism*
  • Synaptic Transmission / physiology*


  • Nerve Tissue Proteins
  • Green Fluorescent Proteins