Computer analysis of double-labeled two-dimensional electrophoresis gels

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Two-dimensional polyacrylamide gel electrophoresis is crucial for separating complex protein mixtures.
• Accurate quantification of protein biosynthesis using double-label autoradiography (3H and 14C) is essential for comparative proteomic studies.

Purpose of the Study:

• To develop and validate a computerized process for the automatic analysis of double-label autoradiography.
• To enable precise and efficient comparison of complex protein mixtures by quantifying isotopic ratios.

Main Methods:

• Digitization of fluorographs and autoradiographs using a rotating drum densitometer.
• Analysis via the Man-computer Interactive Data Analysis System III, employing edge-detection algorithms.
• Conversion of spot density to isotopic disintegrations using standard curves to compute 3H:14C ratios.

Main Results:

• The system accurately locates corresponding protein spots and calculates 3H:14C ratios.
• Average accuracy of calculated ratios is approximately 9% for test samples.
• Analysis of two-dimensional gels demonstrated high precision, with measured ratios closely matching known values.

Conclusions:

• The developed computer process significantly reduces analysis time for comparing protein mixtures.
• The system possesses sufficient precision to detect significant changes in individual protein biosynthesis, such as doubling.