Proteomics/Protein Separations- Electrophoresis/One Dimentional Gel Electrophoresis

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  1. Gel Electrophoresis
  2. One Dimentional Gel Electrophoresis
  3. Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)
  4. Differential in Gel Electrophoresis(DIGE)
  6. Capillary Electrophoresis
  7. Timelines of Electrophoresis
  8. Databases
  9. Web Pages
  10. Online Applications
  11. Further Readings

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)[edit]

This is a very common method of gel 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.The proteins are dissolved in sodium dodecyl sulfate (SDS),a detergent that breaks up the interactions between proteins,and then electrophorised. The smallest molecules move through the gel faster, while larger molecules take longer and result in bands closer to the top of the gel.

Picture of an SDS-PAGE. The molecular marker is in the left lane


The gel used for SDS-PAGE is made out of acrylamide which form crooss-linked polymers of polyacrylamide. Standard gels are typically composed of two layers, one top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and has low pH , while the acrylamide concentration of the separating gel varies according to the samples to be run and has higher pH.The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel.


The samples are treated with SDS (sodium dodecyl sulfate),an anionic detergent which denatures the protein by breaking the disulfide bonds and gives negative charge to each protein in proportion to its mass.Without SDS,different proteins with similar molecular weights would migrate differently due to differences in folding,as differences in folding patterns would cause some proteins to better fit through the gel matrix than others.SDS linearizes the proteins so that they may be separated strictly by molecular weight.The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein [[1]],giving an approximately uniform mass:charge ratio for most proteins,so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.Proteins may be further treated with reducing agent, such as dithiothreitol(DTT) or TRP(Tributyl phosphine)to break any reformed disulfide bonds and then alkalated with iodoacetamide to prevent reformation of disulfide bonds. A tracking dye like bromophenol blue may be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run.


The proteins denatured by SDS are applied to one end of a layer of polyacrylamide gel submerged in a buffer.Buffer provide uniform pH and ions for conducting electric potential. When an electric current is applied across the gel,the negatively-charged proteins migrate across the gel to the positive pole. Short proteins will more easily fit through the pores in the gel and move fast, while larger ones will have more difficulty.Due to differential migration based on their size, smaller proteins move farther down the gel, while larger ones stay closer to the point of origin.After a given period of time,proteins might have separated roughly according to their sizes.Proteins of known molecular weight (marker proteins)can be run in a separate lane in the gel to calibrate the gel.

Staining and analysis[edit]

Following electrophoresis,the gel may be stained with Coomassie Brilliant Blue or silver stain to visualize the separated proteins.After staining, different proteins will appear as distinct bands within the gel accroding to their sizes and thus by molecular weights.The molecular weight of a protein in the band can be estimated by comparing it with the marker proteins of known molecular weights.The separatesd protens can be cut from the gel and further analyzed by other proteomics techniques.

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 run[edit]

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.