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Related Concept Videos

Two-dimensional Gel Electrophoresis01:22

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as  cells...
SDS-PAGE01:27

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
DNA Agarose Gel Electrophoresis02:35

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...

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Related Experiment Video

Updated: Jun 23, 2026

Detection of Protease Activity by Fluorescent Peptide Zymography
09:56

Detection of Protease Activity by Fluorescent Peptide Zymography

Published on: January 20, 2019

Active protease mapping in 2DE gels.

Zhenjun Zhao1, Pamela J Russell

  • 1Institute for Eye Research and Vision Cooperative Research Centre, University of New South Wales, Sydney, NSW, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|April 22, 2009
PubMed
Summary
This summary is machine-generated.

This method isolates and identifies specific proteases in biological samples using 2DE gels and a fluorescent substrate. It allows for characterization of protease activity in its active form.

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Last Updated: Jun 23, 2026

Detection of Protease Activity by Fluorescent Peptide Zymography
09:56

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Published on: January 20, 2019

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling
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09:57

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Published on: December 17, 2016

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Proteases are crucial enzymes mediating essential biological functions.
  • Characterizing proteases in their active state is vital for understanding their roles.
  • Existing methods may not preserve protease activity during separation.

Purpose of the Study:

  • To present a novel method for separating and identifying active proteases.
  • To enable the characterization of individual protease functions within complex biological samples.
  • To visualize protease activity directly within a 2D gel electrophoresis (2DE) system.

Main Methods:

  • Proteases are separated using 2DE under nonreducing conditions.
  • A fluorescently tagged substrate is copolymerized into the second-dimension SDS gel.
  • Proteins are renatured post-electrophoresis to restore protease activity.
  • In situ substrate hydrolysis by active proteases releases fluorescent dye for detection.

Main Results:

  • Specific fluorescent spots pinpoint the location of active proteases in the gel.
  • The method allows for the identification of protease specificity based on substrate cleavage.
  • Enables simultaneous separation and activity-based detection of multiple proteases.

Conclusions:

  • This technique provides a powerful tool for analyzing protease activity and specificity.
  • It facilitates the study of protease function in native, biologically relevant contexts.
  • The method enhances the understanding of protease roles in biological processes.