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Quantitative Analysis of Chromatin Proteomes in Disease
Published on: December 28, 2012
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Protein quantification in complex samples using gel electrophoresis with online intrinsic fluorescence imaging and
Xinke Zhai1, Yiren Cao2, Youli Tian2
1School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China. xuxu3426@sina.com.
Summary
This study introduces an improved gel electrophoresis tank and Gaussian fitting analysis for more accurate and efficient protein quantification. The new method enhances lane capacity and quantitation accuracy in complex samples.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Traditional polyacrylamide gel electrophoresis (PAGE) with staining for protein quantification (PQ) is inaccurate, time-consuming, and complex.
- While intrinsic fluorescence imaging (IFI) improved PAGE, it had limitations in detection window (8 lanes) and quantitation due to low resolution.
Purpose of the Study:
- To enhance gel electrophoresis tank (GET) for a 10-lane detection window with uniform UV radiation.
- To develop a Gaussian fitting arithmetic for accurate protein quantification at low resolutions.
- To improve the utility of PAGE-IFI for analyzing complex protein samples.
Main Methods:
- Modification of the gel electrophoresis tank (GET) to accommodate 10 lanes and ensure uniform UV radiation.
- Development of a Gaussian fitting arithmetic algorithm for precise peak area computation from low-resolution data.
- Application of the improved PAGE-IFI system using bovine serum albumin (BSA) in whey samples for validation.
Main Results:
- The enhanced IFI system enabled simultaneous real-time imaging analysis of 10 lanes.
- The Gaussian fitting analyzer significantly improved peak area computation accuracy for low-resolution data.
- The method demonstrated a low limit of detection (14 ng), limit of quantification (42 ng), good dynamic range (50-8000 ng), and acceptable recovery rates (95-106%) and precision (RSD < 11%).
Conclusions:
- The improved GET and Gaussian fitting analyzer enhance PAGE-IFI for accurate protein analysis in complex samples.
- The developed method offers a more efficient and reliable approach for protein quantification compared to traditional staining methods.
- This advancement holds significant potential for protein determination in diverse biological and food matrices.
