Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein--DNA binding constant

Abstract

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.

SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

“…For gels imaged by methods with linear response (e.g., phosphor storage imaging for 32 P-labeled RNA), quantified gel band intensities corresponding to each nucleotide are extracted for a probed molecule and can be used to determine the midpoints and cooperativity of thermodynamic titrations (Johnson et al 1979;Ackers et al 1982;Celander andCech 1990, 1991), the timescales for folding events (Sclavi et al 1997(Sclavi et al , 1998a, and the changes in solvent accessibility upon folding or binding (Latham and Cech 1989;Felden et al 1994Felden et al , 1996Loizos 2004). Footprinting probes such as the hydroxyl radical (Shadle et al 1997) coupled to gel electrophoresis report information for every nucleotide within a molecule with up to many hundreds of residues in length. Thus, even a single gel can generate thousands of data points.…”

Sequence Discrimination by DNA-binding Domain of ETS Family Transcription Factor PU.1 Is Linked to Specific Hydration of Protein-DNA Interface

“…Electrophoretic data were digitized using a Storm 860 instrument (GE Healthcare). For phosphorimaging data, lane traces were fitted to a superposition of Lorenztian functions without base line (19). Stained gels (excitation/emission ϭ 450/520 nm) were fitted with Gaussian peaks on a heuristic base line to account for background staining.…”

Quantitative Analysis of Electrophoresis Data - Application to Sequence-Specific Ultrasonic Cleavage of DNA

“…This software was exploited to align the gel lanes, calculate the overall intensity of each band and correlate the band sequence with the corresponding nucleotide sequence. The calculation of overall band intensities produced by SAFA is based on several models (Shadle et al, 1997;Takamoto, et al, 2004) introduced earlier to account for such effects as band overlapping and asymmetric distribution of single band intensity along the gel which is better fit with Lorentz function rather than the Gaussian function.…”