"The method makes possible accurate measurements of proteins inside cells using microscopic methods usually used just to show where proteins are located," said senior author Thomas D. Pollard, M.D., Chair and Higgins Professor of Molecular, Cellular & Developmental Biology at Yale, of the work published in Science.

Postdoctoral fellow Jian-Qiu Wu attached a tag called yellow fluorescent protein to proteins of interest, allowing these proteins to be detected in live yeast cells with a light microscope. He used seven sample proteins to demonstrate that the brightness of the fluorescence is directly correlated with the amount of that protein in the cell.

With this reference, they could take a stack of pictures through any whole cell that makes a tagged protein, count up all the fluorescent signal, and calculate the number of molecules by comparing with their standardized sample proteins. The assay works whether the molecules are spread out or concentrated in particular parts of the cell, so they could also count the number of molecules in different locations throughout the cell.

Wu assayed the overall and local concentrations of more than two-dozen proteins. Some regulatory proteins had only a few hundred copies, while the common protein actin had millions of copies. The measurements revealed for the first time the ratios of proteins making up structures such as the "contractile ring" that pinches cells apart as they divide.

"People working on yeast should be able to use this method straight out," said Pollar
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Contact: Janet Rettig Emanuel
janet.emanuel@yale.edu
203-432-2157
Yale University
20-Oct-2005