A laboratory exercise for visible gel filtration chromatography using fluorescent proteins

Area of Science:

• Biochemistry
• Chromatography
• Molecular Biology

Background:

• Gel filtration chromatography (GFC) is a key technique for protein purification, separating molecules based on size.
• Fluorescent proteins offer visual markers for tracking molecular separation.
• Laboratory exercises are crucial for intuitive understanding of complex biochemical techniques.

Purpose of the Study:

• To demonstrate the principles of GFC using prokaryotically expressed fluorescent proteins.
• To provide an intuitive laboratory exercise for students to learn GFC.
• To illustrate factors affecting chromatographic resolution.

Main Methods:

• Prokaryotic expression and purification of red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and their fusion proteins.
• Separation of proteins using Superdex 200 column chromatography.
• Analysis of resolution changes with varying sample volumes and chromatographic resins (Sephadex G-100).

Main Results:

• Distinct bands for RFP, RFP-CFP (RC), YFP-RFP-YFP (YRY), and pyruvate kinase II-GFP (PKG) were well resolved on a Superdex 200 column.
• Increased sample volume and use of Sephadex G-100 resin led to reduced separation resolution.
• Students successfully identified protein combinations and grasped GFC principles.

Conclusions:

• Fluorescent protein fusions are effective tools for demonstrating GFC principles in a laboratory setting.
• Sample volume and resin choice significantly impact GFC resolution.
• This hands-on exercise enhances student comprehension of protein purification techniques.