Specific Protein Quantification by Radioimmuno-Dot-Blot Assay for Complex Mixture Samples Utilizing Strep-Tag and Tritium-Labeled Strep-Tactin

Abstract

Accurately quantifying specific proteins from complex mixtures like cell lysates, for example, during in vivo studies, is difficult, especially for aggregation-prone proteins. Herein, we describe the development of a specific protein quantification method that combines a solid-state dot blot approach with radiolabel detection via liquid scintillation counting. The specific detection with high sensitivity is achieved by using the Twin-Strep protein affinity tag and tritium-labeled ^3HStrep-TactinXT probe. While the assay was developed with the recombinant silk protein CBM-AQ12-CBM as a target, the method can be adapted to other recombinant proteins. Variations of the protein tag and Strep-Tactin probe were tested, and it was found that only the combination of Strep-TactinXT and Twin-Strep-tag performed adequately: with this combination, a precision of 95% and an accuracy of 86% were achieved with a linear region from 19 to 400 ng and a limit of quantification at 0.4 pmol. To achieve this, critical optimization steps were preventing nonspecific adsorption and promoting surface adhesion of the target protein to the solid nitrocellulose membrane. The often-overlooked challenges of sample preparation and protein immobilization in quantification assays are discussed and insights into overcoming such issues are provided.