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Proceedings Paper

Probing protein: DNA interactions using a uniform monolayer of DNA and surface plasmon resonance
Author(s): Jennifer S. Shumaker-Parry; Charles T. Campbell; Gary D. Stormo; Fauzi S. Silbaq; Rudolf H. Aebersold
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Paper Abstract

A method is described for immobilizing double-stranded DNAs to a planar gold surface with high density and uniform spacing. This is accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin. This streptavidin monolayer, which offers approximately 5 X 1012 biotin sites per cm2, is prepared first by adsorbing it onto a mixed self-assembled monolayer on gold which contains biotin-terminated and oligo-terminated alkylthiolates in a 3/7 ratio. This DNA- functionalized surface resists non-specific protein adsorption and is useful for probing the kinetics and equilibrium binding of proteins to DNA with surface plasmon resonance. This is demonstrated with the Mnt protein, which is found to bind in 3.8:1 ratio to its immobilized DNA operator sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA. A sequence with a single base-pair mutation shows nearly as much Mnt binding, but a completely random DNA sequence shows only 5 percent of this binding. This proves that DNA-binding proteins bind sequence-specifically to double-stranded DNAs which are immobilized to gold with this streptavidin linker layer.

Paper Details

Date Published: 21 April 2000
PDF: 9 pages
Proc. SPIE 3922, Scanning and Force Microscopies for Biomedical Applications II, (21 April 2000); doi: 10.1117/12.383343
Show Author Affiliations
Jennifer S. Shumaker-Parry, Univ. of Washington (United States)
Charles T. Campbell, Univ. of Washington (United States)
Gary D. Stormo, Washington Univ. Medical School (United States)
Fauzi S. Silbaq, Univ. of Colorado/Boulder (United States)
Rudolf H. Aebersold, Univ. of Washington (United States)

Published in SPIE Proceedings Vol. 3922:
Scanning and Force Microscopies for Biomedical Applications II
Shuming Nie; Eiichi Tamiya; Edward S. Yeung, Editor(s)

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