A Novel Immobilization Method for Single Protein spFRET Studies
Prithwish Pal 1*, John F Lesoine 1, M Andreas Lieb 1, Lukas Novotny 1 and Philip A Knauf 1
1 University of Rochester
* To whom correspondence should be addressed. E-mail: ppal{at}med.unc.edu.
Submitted on March 11, 2005
Revised on March 15, 2005
Accepted on 28 March 2005
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Abstract |
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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 to 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.
Key Words:
GlpT, Single Molecule Flourescence, cdAE1, protein L, protein immobilization, spFRET
