Quantitative analysis of gel electrophoretograms by image analysis and least squares modeling

Abstract

A computer-aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane-parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gausian curve and an asymmetrical Gauss-Cauchy function. Linear response to the serial dilution of the protein sample is tested.

Kinetics and Diagnostic and Prognostic Potential of Quantitative Western Blot Analysis and Antigen-Specific Enzyme-Linked Immunosorbent Assay in Experimental Canine Leishmaniasis

“…These settings were maintained throughout the analysis. Quantitative analysis of overlapping bands was approximated by fitting the data to a symmetric Gaussian curve (32). Net band intensities were defined as the sum of pixels within the area of the band limited by a preformed rectangular area after subtraction of the background pixels.…”

A simple mathematical model to aid quantification of electrophoresis gels by image analysis

“…Quantities measured included total intensity, average intensity/pixel, absorbance, simple gray levels, average gray scales of electrophoresis bands [7], relative light units and optical density. This quantification has been aided by several pieces of software including NIH Image [7], IBAS 2.0 [8], Image Calc [2] and several systems made from homemade components (see, e.g., [9,[12][13][14][15]). The methods which depend on the capture of images on film, however, require further mathematical work to account for the characteristics of the photographic process itself in relating the values obtained to X-ray intensity or chemical concentrations on the electrophoresis gel.…”

“…The set of Eqs. (9) has been studied extensively over the years, and approximate solutions derived under various conditions. The best known analysis is probably due to Michaelis-Menten (MM) [15,16] which is based upon the standard quasi-steady state approximation (sQSSA [15].…”

A Software System for Data Analysis in Automated DNA Sequencing

“…For DNA separations in polyacrylamide gel electrophoresis, a reasonable approximation of the zonebroadening function is a symmetrical Gaussian (Vohradský and Pánek 1993), in which the width of the Gaussian is the parameter that varies with sample number. In this case the matrix M has the elements…”