Using SDS-PAGE and Scanning Laser Densitometry to Measure Yield and Degradation of Proteins

Abstract

A variety of techniques are available for the quantification of proteins, their degradation products and other impurities. Densitometry is particularly useful due to its sensitivity, accuracy and versatility, and it can be applied to proteins in gels or on membranes and used with numerous detection methods. In addition to being accurate, sensitive and reproducible, the technique is cost-effective, simple, and does not require a high degree of specialized training, yet provides technical advantages over other available tools. For example, Vuletich and Osawa showed that densitometry following SDS-PAGE and electroblotting was 20-fold more sensitive than HPLC at detecting oxidatively modified myoglobin (1). In addition, Morçöl and Subramanian reported the development of a sensitive Ponseau-S-stained dot blot/densitometric protein assay that was less subject to interference by pH, detergent and other reagents than the commonly used Bradford protein assay (2). More recently, Zhu et al. and Miles et al. reported a quantitative densitometric method that measures host cell derived impurity levels on immunoblots of recombinant proteins expressed in E. coli (3, 4). This method was shown to be at least 20 times more sensitive than a commercially available ELISA kit designed to measure host cell impurity levels (3). Other groups have reported on the utility of gel or fluorograph densitometry, using multiple detection methods, in carbohydrate analysis (5), taxonomic and forensic applications (6), as a clinical diagnostic tool in Balkan nephropathy (7) and in the analysis of high density lipoproteins (8), as a means of quantifying relative levels of excretory-secretory polypeptides synthesized in vitro by Schistosoma mansoni daughter sporocysts (9), and for analyzing adsorption of proteins and an oligodeoxynucle-otide in Alhydrogel®-based malaria vaccine candidates (10).