A methodical approach for improving the reliability of quantifiable two-dimensional Western blots

Abstract

Western transfer after the electrophoretic separation of proteins onto an adsorbent membrane, with subsequent immunodetection, is a powerful tool for detecting and characterizing a multitude of proteins. An important aspect of the study of proteins is that they often exist as isoforms with structural microheterogeneity giving rise to differences in biological activity. Western blotting (WB) in combination with two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) allows the specific quantification of single isoforms of a protein. We have investigated whether a methodical modification of 2D-SDS-PAGE improves the quality of quantifiable 2D-WB data. The effect of a combined separation of three previously electrofocused protein extracts lying side by side on a single SDS-gel in parallel has been tested against the traditional procedure, viz., the separation of one protein extract per SDS-gel. The modified procedure results in a more reliable and better quality data than the traditional procedure, which seems to be prone to producing systematic or random errors. Our simple practical procedure improves immunoblotting accuracy by excluding numerous sources of errors and saves time, immunoblotting reagents and costly antibodies.