Comparison of Protein Quantitation Methods Using the Experion Automated Electrophoresis System

Introduction Protein quantitation is a routine analysis procedure required for drug discovery, process development, product manufacturing, and quality control in the pharmaceutical and biopharmaceutical industries. The Experion automated electrophoresis system integrates protein quantitation into a single process in which protein separation, staining, band detection, and quantitation are automatically executed with little user intervention. Compared to conventional SDS-PAGE methods that use gel image analysis for quantitation, the automatic protein quantitation performed by Experion software significantly reduces the labor and time spent in data analysis. However, to use the Experion system as a quantitative tool for protein analysis, it is essential to fully understand and appropriately utilize the protein quantitation techniques provided in Experion software. Experion software offers two different types of protein quantitation methods: percentage determination and concentration determination (Table 1). Percentage determination measures the percentage of each protein in a protein mixture and is commonly used for determining protein content, protein purity, and protein stability, and for checking for mutations. Concentration determination provides the amount of the protein(s) in a protein mixture rather than just a percentage of the total. There are several different ways that concentration determination can be performed, and these methods provide more or less precision depending on the type of internal standard used and the extent to which a calibration curve is used. The ability of the Experion system to provide these different methods of quantitation offers the user the flexibility to customize their quantitation experiments to meet their needs for both throughput and accuracy. This article describes the percentage determination and concentration determination methods used by Experion software, the ways in which these methods are used to achieve different levels of accuracy in protein quantitation, and the considerations that must be made in selecting one method over another.