Proteomics/Protein Separations- Electrophoresis/Types of Gel Electrophoresis

Types of Gel ElectrophoresisEdit

Gel Electrophoresis is a separation technique that is used to separate macromolecules such as nucleic acids or proteins on the basis of size, electric charge, and other physical properties.It can be performed within one dimension(SDS-PAGE,IEF,Native -PAGE), two dimensions(2D-PAGE), or in a capillary. Several forms of PAGE exist and can provide different types of information about the protein(s). Non denaturing PAGE, also called native PAGE, separates proteins according to their mass:charge ratio. SDS-PAGE, the most widely used electrophoresis technique, separates proteins primarily by mass. Two-dimensional PAGE (2D-PAGE) separates proteins by isoelectric point in the first dimension and by mass in the second dimension.


Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)Edit

This is a very common method of elctrophoresis for separating proteins by mass. The most commonly used system is also called the Laemmli method after U.K. Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study.For more information about this technique click on the link.[[1]]

2.Isoelectric Focusing(IEF)Edit

In Isoelectric focusing, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). A pH gradient is generated in the gel and an electric potential is applied across the gel. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.

Immobilized pH Gradeint (IPG) and IEF runEdit

Immobilized pH gradients are used for IEF because the fixed pH gradients remain stable over extended run times at very high voltages. The pH gradients of IPGs are generated by means of buffering compounds that are covalently bound into polyacrylamide gels. IPGs are cast strips with plastic backing sheets and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. Isoelectric focusing is run in the same solutions that are used to extract or solubalize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during which protein samples are loaded into the strips. Rehydration can be active or passive. To load larger proteins active rehydration in small voltage is applied.As the sample is electrophorised, the proteins will migrate toward either the anode or cathode, depending on charge. The proteins will stop when they reach their respective pI. After the run in IEF cell, the proteins focus as bands on the strip according to their isoelectric points. The focused strips can be frozen for storage.


Native PAGE is used to separate proteins in their native states according to difference in their charge density. Native state of protein means proteins are in properly folded state, not denatured or unfolded state. There are no denaturants present in the gel and buffer in the gel maintains the protein in its native state. Many proteins are shown to be enzymatically active after separation by native PAGE. Thus, it is used for prepartion of purified and active proteins. In native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. The charge depend on the amino acid composition of the protein as well as post-translational modifications. The hydrodynamic size and mobility of native protein on the gel will vary with the nature of the conformation. Proteins with compact conformations have higher mobility and larger structures like oligomers have lower mobility. Native PAGE can be carried out near neutral pH to avoid acid or alkaline denaturation to study conformation as well as self-association or aggregation, and the binding of other proteins or compounds. The apparatus is kept cool to minimize denaturation of proteins and proteolysis.

Two Dimensional Polyacrylamide gel Electrophoresis(2D-PAGE)Edit

2D Electrophoresis is a technique that blends the two methods listed in One-Dimensional analyses. It is a very efficient separation technique for proteins. The protein sample is first run on an IPG strip, which after reaching completion is placed in either a horizontal or vertical SDS gel. This technique results in gels that contain spots. Each spot on the gel corresponds to a different protein. Then the gels are stained similarly as to 1D analysis. 2D Electrophoresis is widely used, and certain methods of it can be coupled with mass spectrometry in order to identify proteins.For details about the technique click on the link.[[2]]

Differential in Gel Electrophoresis(DIGE)Edit

Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This technology allows for simultaneous separation and comparison of up to three samples on one gel.For details about the technique click on the link.[[3]]


Quantitative Preparative Native Continuous PolyAcrylamide GelElectrophoresis is a technique to isolate active or native metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. For details about the technique click on the link.[[4]]


Last modified on 14 August 2007, at 06:45