Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell[1] and Klose[2] in 1975.
Basis for separation
2-D electrophoresis begins with electrophoresis in the first dimension and then separates the molecules perpendicularly from the first to create an electropherogram in the second dimension. In electrophoresis in the first dimension, molecules are separated linearly according to their isoelectric point. In the second dimension, the molecules are then separated at 90 degrees from the first electropherogram according to molecular mass. Since it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.
The two dimensions that proteins are separated into using this technique can be
- The separation by isoelectric point is called isoelectric focusing. Thereby, a pH gradient is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pH values other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).
- Separation by protein complex mass is done via native PAGE, in which proteins remain in their native state and are separated in the electric field following their mass and the mass of their complexes respectively. To obtain a separation by size and not by net charge, as in IEF, an additional charge is transferred to the proteins by the use of Coomassie brilliant blue or lithium dodecyl sulfate. Knowledge of protein complex is important for the analysis of the functioning of proteins in a cell, as proteins mostly act together in complexes to be fully functional. The analysis of this sub organelle organisation of the cell requires techniques conserving the native state of the protein complexes.
- Separate just by mass is commonly achived using SDS-PAGE. SDS denatures the proteins, breaks apart most complexes, and approximately equalizes the mass-to-charge ratios. SDS must be done as the second, perpendicular dimension, as it breaks apart complexes (rendering native PAGE impossible) and equalizes mass-to-charge ratios (rendering IEF impossible).
Detecting proteins
The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are
Molecules other than proteins can be separated by 2D electrophoresis. In
Common techniques
This section may require cleanup to meet Wikipedia's quality standards. The specific problem is: Wait a second, isoelectric focusing also uses an IPG per its article, and it is also followed by SDS. What's the real difference, if any? (December 2023) |
IPG-DALT
A common technique is to use an Immobilized pH gradient (IPG) in the first dimension. This technique is referred to as IPG-DALT. The sample is first separated onto IPG gel (which is commercially available) then the gel is cut into slices for each sample which is then equilibrated in SDS-mercaptoethanol and applied to an SDS-PAGE gel for resolution in the second dimension. Typically IPG-DALT is not used for quantification of proteins due to the loss of low molecular weight components during the transfer to the SDS-PAGE gel.[4]
IEF SDS-PAGE
2D gel analysis software
In quantitative proteomics, these tools primarily analyze bio-markers by quantifying individual proteins, and showing the separation between one or more protein "spots" on a scanned image of a 2-DE gel. Additionally, these tools match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. Software packages include Delta2D (discontinued), ImageMaster (discontinued), Melanie, PDQuest (discontinued), SameSpots and REDFIN – among others.[citation needed] While this technology is widely utilized, the intelligence has not been perfected. For example, while PDQuest and SameSpots tend to agree on the quantification and analysis of well-defined well-separated protein spots, they deliver different results and analysis tendencies with less-defined less-separated spots.[5] Comparative studies have previously been published to guide researchers on the "best" software for their analysis.[6] Although typically used for standard gel electrophoresis, Sciugo can also be used for figure-creation and quantification.
Challenges for automatic software-based analysis include incompletely separated (overlapping) spots (less-defined or separated), weak spots / noise (e.g., "ghost spots"), running differences between gels (e.g., protein migrates to different positions on different gels), unmatched/undetected spots, leading to
mismatched spots, errors in quantification (several distinct spots may be erroneously detected as a single spot by the software and parts of a spot may be excluded from quantification), and differences in software algorithms and therefore analysis tendenciesGenerated picking lists can be used for the automated in-gel digestion of protein spots, and subsequent identification of the proteins by mass spectrometry. Mass spectrometry analysis can identify precise mass measurements along with the sequencing of peptides that range from 1000–4000 atomic mass units. [9] For an overview of the current approach for software analysis of 2DE gel images, see Berth et al.[10] or Bandow et al.[11]
See also
- Difference gel electrophoresis
- PROTOMAP
References
- PMID 236308.
- S2CID 30981877.
- PMID 94518.
- ISBN 978-0-471-62386-1.
- S2CID 45193214.
- PMID 19133722.
- S2CID 21152053.
- ^ What are missing values, and why are they a problem?
- ^ Lepedda, Antonio J, and Marilena Formato. "Applications of Two-Dimensional Electrophoresis Technology to the Study of Atherosclerosis." EJIFCC vol. 19,3 146–159. 20 Dec. 2008
- PMID 17713763.
- S2CID 206361897.
External links
- JVirGel Create virtual 2-D Gels from sequence data.
- Gel IQ A freely downloadable software tool for assessing the quality of 2D gel image analysis data.
- 2-D Electrophoresis Principles & Methods Handbook