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Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

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Single-molecule tracking of carbohydrate-binding modules on cellulose using fluorescence microscopy.

Yu-San Liu¹, Shi-You Ding, Michael E Himmel

¹National Renewable Energy Laboratory, Biosciences Center, Golden, CO, USA. yu.san.liu@nrel.gov

Methods in Molecular Biology (Clifton, N.J.)

|July 31, 2012

Summary

This chapter details Total Internal Reflection Fluorescence Microscopy (TIRF-M) protocols for visualizing single carbohydrate-binding module (CBM) molecules labeled with green fluorescent protein (GFP). These methods enable the study of molecular interactions in biological systems.

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Area of Science:

Biophysics
Molecular Biology
Microscopy

Background:

Single-molecule fluorescence detection is crucial for understanding molecular behavior in biological systems.
Carbohydrate-binding modules (CBMs) play vital roles in carbohydrate metabolism and recognition.
Green fluorescent protein (GFP) is a widely used fluorescent marker in biological research.

Purpose of the Study:

To provide detailed protocols for visualizing single CBM-GFP molecules using Total Internal Reflection Fluorescence Microscopy (TIRF-M).
To enable the study of molecular interactions, including cellulase-cellulose interactions, at the single-molecule level.

Main Methods:

Detailed step-by-step protocols for sample preparation for TIRF-M.
Methods for data acquisition, processing, and analysis of single-molecule fluorescence data.
Utilizing TIRF-M to visualize single CBM-GFP molecules.

Main Results:

Successful visualization of single CBM-GFP molecules using TIRF-M.
Established protocols for sample preparation and data analysis.
Demonstrated applicability for studying molecular interactions.

Conclusions:

TIRF-M is a powerful technique for single-molecule studies of CBMs.
The described protocols facilitate the investigation of molecular interactions in biological systems.
These methods can be extended to study cellulase-cellulose interactions.