Protein quantification by means of a stain-free SDS-PAGE technology without the need for analytical standards: Verification and validation of the method

Abstract

Proteins in food systems can be measured quantitatively by SDS-PAGE based on stainability of the protein fractions and only if analytical standard material is available, which is often not the case for minor protein components. In this study, a novel stain-free SDS-PAGE technique was evaluated and validated to see if the tryptophan content in proteins can be used as an absolute quantification criterion. Induced by UV light irradiation, tryptophan residues react with trichloroethanol, which is imbedded in the SDS-PAGE gel and produces fluorescence with variable intensities, depending on the number of tryptophan residues. Our results show good linear correlation between the analyzed fluorescence intensity and the absolute mass of tryptophan per protein across a wide range of different proteins. Successful validation of the method was conducted using model proteins that were treated as if they were proteins without available analytical standards. Calculated protein concentrations based on their respective fluorescence intensities showed no significant deviations in comparison to protein quantification by HPLC analysis. In conclusion, proteins can be determined quantitatively based on the total amount of tryptophan without staining and without the need for analytical standard material, provided that their amino acid composition and distribution is known.