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A Novel Immobilization Method for Single Protein spFRET Studies

Prithwish Pal ^*, John F. Lesoine , M. Andreas Lieb , Lukas Novotny  and Philip A. Knauf ^*

^* Department of Biochemistry and Biophysics, University of Rochester, New York 14642; Institute of Optics, University of Rochester, New York 14642

Correspondence: Address reprint requests and inquiries to Philip A Knauf, Tel.: 585-275-5459; Fax: 585-273-4746; E-mail: philip.knauf{at}rochester.edu.

We have developed a new method for immobilization of single proteins by utilizing streptavidin-biotin and protein L-antibody interactions on glass coverslips coated with polyethylene glycol. The method is particularly well suited for single-molecule fluorescence studies. A monomeric, detergent-solubilized bacterial transport protein, GlpT, and the dimeric cytoplasmic region of a mammalian transporter, cdAE1, were immobilized by our method with a high degree of specificity. The fluorescence from single molecules attached to the immobilized proteins was detected with a high signal/noise ratio. Single-pair fluorescence resonance energy transfer (spFRET) measurements on cdAE1 dimers indicate that the structure of the protein is not compromised and provide evidence that the cdAE1 protein can exist in at least two conformations under physiological conditions.