The article describes a high-resolution two-dimensional polyacrylamide gel electrophoresis technique for protein separation. The method involves isoelectric focusing in the first dimension to separate proteins by their isoelectric points, followed by sodium dodecyl sulfate (SDS) electrophoresis in the second dimension to separate them by molecular weight. This technique provides a powerful tool for analyzing and detecting proteins from complex biological sources, with a resolving power sufficient to distinguish 1100 different components from *Escherichia coli* and the potential to resolve up to 5000 proteins. The technique is sensitive enough to detect proteins containing as little as one disintegration per minute of either ${ }^{14} \mathrm{C}$ or ${ }^{35} \mathrm{~S}$, and proteins representing $10^{-4}$ to $10^{-5} \%$ of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation allows for matching spots on different gels, making it suitable for estimating the number of proteins produced by any biological system. The technique can also be used to identify missense mutations that alter protein charge. The article provides detailed methods for preparing samples, constructing gels, and analyzing results, including the measurement of pH gradients, spot sizes, and standard deviations.The article describes a high-resolution two-dimensional polyacrylamide gel electrophoresis technique for protein separation. The method involves isoelectric focusing in the first dimension to separate proteins by their isoelectric points, followed by sodium dodecyl sulfate (SDS) electrophoresis in the second dimension to separate them by molecular weight. This technique provides a powerful tool for analyzing and detecting proteins from complex biological sources, with a resolving power sufficient to distinguish 1100 different components from *Escherichia coli* and the potential to resolve up to 5000 proteins. The technique is sensitive enough to detect proteins containing as little as one disintegration per minute of either ${ }^{14} \mathrm{C}$ or ${ }^{35} \mathrm{~S}$, and proteins representing $10^{-4}$ to $10^{-5} \%$ of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation allows for matching spots on different gels, making it suitable for estimating the number of proteins produced by any biological system. The technique can also be used to identify missense mutations that alter protein charge. The article provides detailed methods for preparing samples, constructing gels, and analyzing results, including the measurement of pH gradients, spot sizes, and standard deviations.