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Western Blotting

Western Blotting

Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.

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Detection of Apoplastic Protease Inhibitors Using Convolution Activity-Based Protein Profiling.

Andrea Passarge¹, Gunther Doehlemann^1,2, Johana C Misas Villamil^3,4

¹Institute for Plant Sciences, University of Cologne, Cologne, Germany.

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

|May 18, 2022

Summary

Convolution activity-based protein profiling (ABPP) monitored Lolium perenne apoplastic cysteine protease activity during fungal endophyte interaction. This method detects changes in protease inhibitor abundance, offering insights into plant-microbe interactions.

Keywords:

Activity Apoplast Convolution ABPP Endophyte PLCP Proteases Ryegrass

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Last Updated: Sep 22, 2025

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Area of Science:

Biochemistry
Plant Pathology
Molecular Biology

Background:

Activity-based protein profiling (ABPP) is a key technique in chemical biology.
ABPP utilizes chemical probes to covalently label and monitor enzyme activity, particularly proteases.
Existing ABPP methods include comparative, competitive, and convolution approaches.

Purpose of the Study:

To describe and apply the convolution ABPP method for detecting protease inhibitor abundance changes.
To investigate the activity of Lolium perenne apoplastic cysteine proteases during Epichloë festucae infection.
To establish a protocol for analyzing protease activity in plant-microbe interactions.

Main Methods:

Isolation of apoplastic fluids from infected and uninfected L. perenne ryegrass leaves.
Performance of convolution ABPP experiments using specific chemical probes.
Quantification and analysis of fluorescent gels from ABPP labeling.

Main Results:

Demonstration of the convolution ABPP method's utility in monitoring protease activity.
Insights into changes in apoplastic cysteine protease activity in L. perenne upon E. festucae interaction.
Detailed protocols for sample preparation, ABPP, and data analysis.

Conclusions:

Convolution ABPP is effective for studying protease activity in complex biological systems.
The study provides a framework for understanding plant protease responses to fungal endophytes.
This research contributes to the understanding of plant-microbe molecular interactions.