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Characterizing specific phage–protein interactions by fluorescence correlation spectroscopy
Author(s) -
Bahns John T.,
Liu ChinMei,
Chen Liaohai
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04695704
Subject(s) - fluorescence correlation spectroscopy , autocorrelation , fluorescence , fluorescence spectroscopy , chemistry , biological system , biophysics , phage display , spectroscopy , dissociation constant , dissociation (chemistry) , chemical physics , biology , physics , optics , biochemistry , mathematics , peptide , statistics , receptor , quantum mechanics
The interactions of several affinity reagent displayed T7 and M13 phage particles with their corresponding target molecules were examined using Fluorescence Correlation Spectroscopy (FCS). Diffusion times, relative fractions of each component in the recognition reactions at the equilibrium state, and ultimately the dissociation constants were deduced from analyzing the fluorescence autocorrelation curves. Although the sample preparation and FCS characterization of icosahedral T7‐related systems were relatively straight forward, procedures with filamentous M13‐related systems were complicated by the physical size of M13 and its aggregate formation. Methods that accommodate the FCS measurement of the M13 phage via changing confocal optics, fitting procedures, and aggregate discrimination are presented and discussed.