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High-performance affinity chromatography of human serum concanavalin A binding proteins
T Manabe1, N Higuchi, T Okuyama
1Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Japan.
Journal of Chromatography
|September 23, 1988
Summary
A novel concanavalin A (Con A) affinity column effectively fractionated human serum Con A-binding proteins in under 80 minutes. Despite capacity loss, its specificity for Con A-binding proteins remained consistent during repeated use.
Area of Science:
- Biochemistry
- Proteomics
- Affinity Chromatography
Background:
- Concanavalin A (Con A) is a lectin that binds to specific carbohydrate structures on proteins.
- Fractionating Con A-binding proteins from complex biological samples like human serum is crucial for understanding their functions.
Purpose of the Study:
- To evaluate the efficacy of a high-performance Con A affinity column (Gelpack GL-L55C) for fractionating human serum Con A-binding proteins.
- To assess the column's specificity, capacity, and reusability.
Main Methods:
- Utilized a Gelpack GL-L55C Con A affinity column for human serum fractionation.
- Analyzed eluates using micro two-dimensional electrophoresis, blotting, and Con A staining.
- Investigated column capacity changes with repeated loading of serum and tissue extracts.
Main Results:
- Successfully fractionated Con A-binding proteins (approximately 11% of recovered proteins) within 80 minutes.
- Confirmed column specificity through electrophoretic and blotting analyses.
- Observed a gradual decrease in protein-binding capacity with repeated use, attributed to lipid/lipoprotein binding.
Conclusions:
- The Con A affinity column provides an efficient method for isolating specific Con A-binding proteins from human serum.
- The column maintains high specificity for Con A-binding proteins despite capacity reduction over time.
- Lipid or lipoprotein binding likely causes the observed decrease in column capacity.