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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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A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

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Published on: February 16, 2014

Macrocyclic Phage Display for Identification of Selective Protease Substrates.

Franco F Faucher^1,2, Kristýna Blažková², Scott Lovell^2,3

¹Department of Chemistry, Stanford University, Stanford, California 94305, United States.

Journal of the American Chemical Society

|July 18, 2025

Summary

This study introduces a novel phage display method to discover highly selective protease substrates using chemically modified cyclic peptides. This approach overcomes limitations of traditional methods and enables new tools for biology and medicine.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Traditional methods for identifying protease substrates use linear peptides with limited diversity.
Phage display is a powerful tool for screening large peptide libraries.

Purpose of the Study:

To develop a novel phage display approach for identifying highly selective, chemically modified cyclic peptide substrates for proteases.
To overcome limitations of existing methods in terms of sequence and structural diversity.

Main Methods:

Utilizing phage display to screen chemically modified cyclic peptide libraries on the phage surface.
Employing a reactive chemical linker for cyclization, an affinity tag for capture and release, and a turn-on fluorescent reporter for cleavage quantification.
Demonstrating the method using Fibroblast Activation Protein α (FAPα) and dipeptidyl peptidase-4 (DPP4) as target proteases.

Main Results:

Successfully identified highly selective protease substrates for FAPα and DPP4.
Demonstrated the utility of the identified substrates for applications in basic biology and fluorescence image-guided surgery (FIGS).
Validated the rapid and unbiased nature of the platform for discovering non-natural protease substrates.

Conclusions:

The developed phage display strategy offers a rapid, unbiased, and effective platform for discovering highly selective protease substrates.
This method overcomes key limitations of traditional approaches, enabling broader applications in biological research and medical diagnostics.
The identified substrates hold significant potential as valuable tools for FAPα and DPP4 related research and clinical applications.