Nucleic Acids Research, Vol 25, Issue 4 850-860, Copyright © 1997 by Oxford University Press
SE Shadle, DF Allen, H Guo, WK Pogozelski, JS Bashkin and TD Tullius
A computer program, GelExplorer, which uses a new methodology for obtaining
quantitative information about electrophoresis has been developed. It
provides a straightforward, easy-to-use graphical interface, and includes a
number of features which offer significant advantages over existing methods
for quantitative gel analysis. The method uses curve fitting with a
nonlinear least-squares optimization to deconvolute overlapping bands.
Unlike most curve fitting approaches, the data is treated in two
dimensions, fitting all the data across the entire width of the lane. This
allows for accurate determination of the intensities of individual,
overlapping bands, and in particular allows imperfectly shaped bands to be
accurately modeled. Experiments described in this paper demonstrate
empirically that the Lorentzian lineshape reproduces the contours of an
individual gel band and provides a better model than the Gaussian function
for curve fitting of electrophoresis bands. Results from several fitting
applications are presented and a discussion of the sources and magnitudes
of uncertainties in the results is included. Finally, the method is applied
to the quantitative analysis of a hydroxyl radical footprint titration
experiment to obtain the free energy of binding of the lambda repressor
protein to the OR1 operator DNA sequence.
ARTICLES
Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant
Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA.
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