Related Experiment Video
Updated: Jul 22, 2026
08:17
Electrophoretic Separation of Proteins
Published on: June 12, 2008
The role of separation science in proteomics research
1Separation Technology Group, SAIC-Frederick/NCI-Frederick, MD 21702, USA. issaqh@mail.ncifcrf.gov
Electrophoresis
|November 9, 2001
Summary
This review explores protein separation techniques like gel electrophoresis, HPLC, and CE in proteomics. It highlights their capabilities, limitations, and advanced multi-dimensional strategies for protein identification.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Growing interest in comprehensive protein analysis within cells and tissues.
- Need for advanced techniques to separate, quantify, and identify proteins.
Purpose of the Study:
- To review the role of gel electrophoresis, HPLC, and CE in proteomics.
- To compare single and multi-dimensional separation strategies.
- To discuss novel approaches in protein analysis.
Main Methods:
- Review of established separation techniques: gel electrophoresis, HPLC, and CE.
- Analysis of instrumental strategies for protein resolution.
- Examination of multi-dimensional approaches (e.g., 2D-PAGE, HPLC-HPLC, HPLC-CE).
Main Results:
- Capabilities and limitations of individual separation methods are detailed.
- Multi-dimensional separations offer advantages for complex protein mixtures.
- Mass spectrometry is crucial for protein identification post-separation.
Conclusions:
- Efficient protein separation is key to advancing proteomics research.
- Multi-dimensional techniques enhance resolution and identification capabilities.
- Novel approaches continue to emerge for protein concentration, separation, detection, and quantification.
Related Concept Videos
01:20Overview Of Cell Separation And Isolation
Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.
01:32Subcellular Fractionation
The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.
Differential Centrifugation
Differential centrifugation is...
Differential Centrifugation
Differential centrifugation is...
01:22Two-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...
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...
01:33Peptide Identification Using Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
01:33Proteomics
A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics...
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics...
01:08Size-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,...
Silica particles offer advantages such as rigidity,...