Protein quantification in complex samples using gel electrophoresis with online intrinsic fluorescence imaging and Gaussian fitting arithmetic

Abstract

Traditional polyacrylamide gel electrophoresis (PAGE) has been widely used for analysis of proteins. However, protein quantification (PQ) based on SDS-PAGE with staining remains inaccurate, time-consuming and complex. Even though PAGE with online intrinsic fluorescence imaging (IFI) has well addressed these issues, it still suffers from the narrow detection window of 8 lanes and poor quantitation owing to low resolution. Herein, we improved the gel electrophoresis tank (GET) for a wider detection window of 10 lanes with uniform UV radiation and developed a Gaussian fitting arithmetic for accurate PQ at low resolutions. To demonstrate the improvement, we used bovine serum albumin (BSA) in whey samples as a model protein and conducted the relevant experiments. As demonstrated by the experiments, the IFI system could be well used for the simultaneous real-time imaging analysis of 10 lanes. In particular, the Gaussian fitting-based analyzer could be used for the computation of peak area with low resolution, greatly improving the function of PAGE-IFI for protein analysis of complex samples. In addition, the developed method exhibited a good limit of detection (LOD) of 14 ng and limit of quantification (LOQ) of 42 ng, a fair dynamic range of 50–8000 ng, recovery of 106.37% for a urine sample, recovery of 94.96% for the whey sample, intra-day RSD value of 9.17%, and inter-day RSD value of 10.06%. All these results indicated the potential of the developed GET, Gaussian fitting-based analyzer and the proposed method for the determination of protein in complex samples.