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Two-dimensional polyacrylamide gel electrophoresis
8/1/03. By Richard Twyman
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a technique for separating complex mixtures of proteins.
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Thousands of proteins can be resolved in a single experiment allowing the major proteins in a sample to be isolated and protein levels in related samples to be compared. In combination with mass spectrometry, the proteins can also be identified
Key principles
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Proteins differ from each other in terms of their mass and charge.
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Both these properties can be used to separate proteins by gel electrophoresis.
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The successive application of both techniques in perpendicular directions (two dimensions) provides maximum separation and allows thousands of proteins to be resolved.
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Staining the gel reveals the positions of individual proteins as spots or smudges. These can be picked and analysed by mass spectrometry.
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There are tens of thousands of proteins in the cell, differing in abundance over six orders of magnitude. 2D-PAGE is not sensitive enough to detect the rare proteins and many proteins will not be resolved. Splitting the sample into different fractions is often necessary to reduce the complexity of protein mixtures prior to 2D-PAGE.
How does it work?
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Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is used to separate mixtures of proteins, and is particularly useful for comparing related samples - such as healthy and diseased tissue. The first step is to load the proteins onto the gel, which has a pH gradient from top to bottom.
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When a voltage is applied across the gel, the proteins move through the gel until they reach the point at which their charge is the same as the surrounding pH. This separation is called isoelectric focusing.
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Having separated the proteins by charge, the second step is to separate them according to their mass in the perpendicular dimension. Individual proteins can then by isolated and identified by mass spectrometry. Comparing the two gels can reveal proteins that are expressed at different levels. For example, the red protein is more abundant in the diseased tissue, and could be
a useful drug target.
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The polyacrylamide gel provides a supporting matrix through which proteins can migrate. This gel has a pH gradient from top to bottom - the top is more acidic than the bottom.
A complex protein mixture is loaded in the middle of the left side, where the pH is neutral, and a voltage is applied across the gel. The proteins then migrate through the gel until they reach their isoelectric point (the point at which their charge is the same as the surrounding pH). This technique is called isoelectric focussing.
The gel is then soaked in a denaturing solution (which causes the proteins to unfold) containing the detergent sodium dodecylsulphate (SDS). This molecule has a very strong negative charge and binds to all proteins, effectively making them all the same charge.
A voltage is then applied across the gel from side to side with the anode (positive terminal) at the right. The proteins all move towards the anode, but smaller proteins move faster through the gel than larger ones, thus separating the proteins according to their size.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is used to separate mixtures of proteins, and is particularly useful for comparing related samples Ð such as healthy and diseased tissue. The first step is to load the proteins onto the gel, which has a pH gradient from top to bottom.
How is it used?
2D-PAGE can be used in combination with mass spectrometry to systematically identify all the major proteins present in a sample.
However, the technique is particularly powerful when comparing related samples, such as healthy tissue vs disease tissue. For example, proteins that are more abundant in disease tissue may represent novel drug targets or diagnostic disease markers.
Comparative 2D-PAGE can also be used to look for proteins whose expression varies similarly under the same set of conditions (these may have related functions) and to identify proteins produced in response to drug therapy (these may be responsible for drug-related side effects).
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