Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells.

Jacek T Mika, Victor Krasnikov, Geert van den Bogaart, Foppe de Haan, Bert Poolman

Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.

PloS one 2011

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Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP). Throughout literature a wide range of diffusion coefficients for GFP in the cytoplasm of Escherichia coli (3 to 14 µm²/s) is reported using FRAP-based approaches. In this study, we have evaluated two of these methods: pulsed-FRAP and "conventional"-FRAP. To address the question whether the apparent discrepancy in the diffusion data stems from methodological differences or biological variation, we have implemented and compared the two techniques on bacteria grown and handled in the same way. The GFP diffusion coefficients obtained under normal osmotic conditions and upon osmotic upshift were very similar for the different techniques. Our analyses indicate that the wide range of values reported for the diffusion coefficient of GFP in live cells are due to experimental conditions and/or biological variation rather than methodological differences.

Citation

Jacek T Mika, Victor Krasnikov, Geert van den Bogaart, Foppe de Haan, Bert Poolman. Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells. PloS one. 2011;6(9):e25664