Protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach.

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Traditional centrifugation-based protein-liposome assays have limitations, particularly for spontaneously precipitating proteins.
• These methods also offer restricted capabilities for precise protein quantification.

Purpose of the Study:

• To develop and present a novel fluorescence microscopy-based protocol for comparing relative protein-liposome binding affinity.
• To offer a more versatile alternative to existing centrifugation assays.

Main Methods:

• Preparation of fluorescent liposomes.
• Extraction of proteins from fission yeast.
• Performing the liposome binding assay.
• Confocal imaging and data analysis.

Main Results:

• The developed protocol enables effective comparison of protein-liposome binding affinities.
• The method is suitable for proteins that spontaneously precipitate in traditional assays.
• Quantitative analysis of binding is achievable.

Conclusions:

• This fluorescence microscopy protocol provides a robust method for assessing protein-liposome interactions.
• The protocol is adaptable for non-transmembrane proteins from diverse in vivo and in vitro systems.
• It offers improved quantification and broader applicability compared to centrifugation methods.