Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

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...
Size-Exclusion Chromatography01:08

Size-Exclusion Chromatography

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
Electrophoresis: Overview01:20

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
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...
Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Reliability of the International Spinal Cord Injury Physical Therapy-Occupational Therapy Basic Data Set.

Neurotrauma reports·2024
Same author

Platform Methods to Characterize the Charge Heterogeneity of Three Common Protein Therapeutics by Imaged Capillary Isoelectric Focusing.

Methods in molecular biology (Clifton, N.J.)·2021
Same author

Simple Western: Bringing the Western Blot into the Twenty-First Century.

Methods in molecular biology (Clifton, N.J.)·2021
Same author

Cytomegalovirus vectors expressing Plasmodium knowlesi antigens induce immune responses that delay parasitemia upon sporozoite challenge.

PloS one·2019
Same author

Plasmodium inui infection reduces the efficacy of a simian immunodeficiency virus DNA vaccine in a rhesus macaque model through alteration of the vaccine-induced immune response.

The Journal of infectious diseases·2012
Same author

Extraction and solubilization of total protein from microorganisms.

CSH protocols·2012

Related Experiment Video

Updated: Jul 6, 2026

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
09:57

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Published on: June 14, 2020

Sample prefractionation in granulated sephadex IEF gels.

Angelika Görg1, Carsten Lück, Walter Weiss

  • 1Technical University of Munich, Munich, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|March 29, 2008
PubMed
Summary

We developed a simple, fast, and cost-effective prefractionation method using flat-bed isoelectric focusing (IEF) in Sephadex gels. This technique enhances the detection of low-abundance proteins in two-dimensional electrophoresis (2-DE).

More Related Videos

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery
07:57

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Published on: December 3, 2009

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis
11:38

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Published on: February 29, 2016

Related Experiment Videos

Last Updated: Jul 6, 2026

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
09:57

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Published on: June 14, 2020

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery
07:57

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Published on: December 3, 2009

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis
11:38

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Published on: February 29, 2016

Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Reducing sample complexity is crucial for detecting low-abundance proteins in complex biological samples.
  • Traditional prefractionation methods can be time-consuming, costly, or lead to sample loss.
  • Two-dimensional electrophoresis (2-DE) is a powerful technique for protein separation but can be limited by sample complexity and protein abundance.

Purpose of the Study:

  • To develop a simple, rapid, and economical prefractionation method for enhancing protein detection in 2-DE.
  • To improve the identification of low-abundance proteins from both prokaryotic and eukaryotic samples.
  • To create a prefractionation technique compatible with immobilized pH gradient (IPG) strips for seamless integration into 2-DE workflows.

Main Methods:

  • Developed a prefractionation procedure utilizing flat-bed isoelectric focusing (IEF) in granulated Sephadex gels.
  • Incorporated chaotropes, zwitterionic detergents, and carrier ampholytes into the Sephadex gel matrix for effective protein focusing.
  • Applied Sephadex gel fractions directly onto narrow pH range IPG strips for the first dimension of 2-DE, ensuring efficient electrophoretic transfer without dilution or desalting.

Main Results:

  • The prefractionation method effectively reduced sample complexity, enabling higher protein loading on 2-DE gels.
  • Achieved highly efficient electrophoretic transfer of proteins from Sephadex IEF fractions to IPG strips.
  • Demonstrated successful application in the separation, detection, and identification of low-abundance proteins from diverse biological samples.

Conclusions:

  • The developed Sephadex gel IEF prefractionation method is a simple, fast, and cost-effective approach for enhancing protein analysis in 2-DE.
  • This technique overcomes limitations of sample complexity and protein abundance, facilitating the discovery of low-abundance proteomes.
  • The method's compatibility with IPG strips and 2-DE workflows makes it a valuable tool for proteomic research.