Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone

Authors: GAI, HONGWEI; GRIESS, GARY A.; DEMELER, BORRIES; WEINTRAUB, SUSAN T.; SERWER, PHILIP

Source: Journal of Microscopy, Volume 226, Number 3, June 2007 , pp. 256-262(7)

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Abstract:

Summary

To bypass limitations of ensemble averaging biochemical analysis, microscopy-based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488-stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re-swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.

Keywords: Agarose; alexa dye; bovine serum albumin; restricted thermal motion; single-molecule biochemistry

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1365-2818.2007.01776.x

Affiliations: 1: Department of Biochemistry The University of Texas Health Science Center 7703 Floyd C Drive San Antonio, Texas 78229-3900, U.S.A

Publication date: 2007-06-01