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Nat Methods. 2005 Sep;2(9):677-84.

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

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1
Department of Cell Biology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.

Abstract

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.

PMID:
16118638
DOI:
10.1038/nmeth783
[Indexed for MEDLINE]
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