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Detection and quantification of biotinylated proteins using the Storm 840 Optical Scanner
Brandon Lewis1, Sara Rathman, Robert J McMahon
1Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences and the, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611-0370, USA.
The Journal of Nutritional Biochemistry
|May 29, 2003
Summary
Avidin-biotin detection systems offer sensitive protein analysis. Avidin-alkaline phosphatase with ECF substrate provided the highest sensitivity for detecting biotinylated polypeptides.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Avidin-biotin interaction is a widely used method for protein detection.
- Fluorescence detection can enhance sensitivity and linearity in avidin-biotin assays.
Purpose of the Study:
- To evaluate three fluorescent systems for detecting biotinylated polypeptides.
- To compare the sensitivity and linearity of different avidin-biotin-fluorescence detection methods.
Main Methods:
- Tested Neutravidin-Alexa Fluor430, avidin-horseradish peroxidase with ECL-Plus, and avidin-alkaline phosphatase with ECF.
- Used SDS-PAGE, Western blotting to PVDF membranes, and Storm840 optical scanner for visualization.
- Analyzed biotinylated molecular weight standards, bovine serum albumin, and rat liver homogenate.
Main Results:
- Neutravidin-Alexa Fluor430 showed high linearity but lower sensitivity.
- Avidin-horseradish peroxidase and avidin-alkaline phosphatase systems exhibited higher fluorescence.
- The avidin-alkaline phosphatase ECF system demonstrated the highest sensitivity.
Conclusions:
- All tested systems can reliably detect and quantify biotinylated polypeptides.
- Avidin-alkaline phosphatase with ECF offers superior sensitivity for biotinylated protein detection.
- Optimization of conditions is crucial for reliable results with each system.