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Quantitative analysis of two-dimensional electrophoretograms.

J Bossinger, M J Miller, K P Vo

    The Journal of Biological Chemistry
    |August 25, 1979
    PubMed
    Summary
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    This study introduces a novel method for quantifying radioactivity in autoradiograms, specifically for two-dimensional gel electrophoresis protein analysis. The technique accurately measures protein distribution and intensity from digitized film density data.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Analytical Chemistry

    Background:

    • Autoradiograms are crucial for visualizing and quantifying radioactive isotopes in biological samples.
    • Analyzing complex film density distributions, especially in two-dimensional gel electrophoresis, presents significant challenges for accurate protein quantification.

    Purpose of the Study:

    • To develop and describe a robust method for the quantitative analysis of complex film density distributions in autoradiograms.
    • To enable precise measurement of radioactivity distribution among proteins resolved by two-dimensional gel electrophoresis.

    Main Methods:

    • Digitization of autoradiogram film density using a high-speed rotating drum scanner.
    • Image data processing including averaging, contour detection, spot splitting, and density-to-radioactivity conversion using calibration films.

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  • Differentiation and integration techniques to quantify total radioactivity per protein spot.

    • Main Results:

    • Generation of a numbered contour map detailing resolved spots.
    • A comprehensive table listing coordinates and radioactivity content for each identified spot.
    • Development of coordinate transformations for comparative autoradiogram analysis.

    Conclusions:

    • The described method provides a quantitative approach for analyzing autoradiogram film density distributions.
    • This technique is particularly suitable for measuring radioactivity in proteins separated by two-dimensional gel electrophoresis.
    • The developed system facilitates accurate quantification and comparison of radioactive signals in complex biological samples.