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2-D Gel Electrophoresis | Analyze ProteinsBY: Muniba Safdar | Category: Genetics | Submitted: 2010-07-31 06:09:48
Article Summary: "What is 2-D gel electrophoresis? The 2-D gel electrophoresis is abbreviated as two dimensional or 2-DE. It is a type of gel electrophoresis (that separates molecules on the basis of their rate of movement through a gel). It is usually used to analyze proteins. Proteins are separated on the basis of two properties by mass and cha.."
2-D gel electrophoresis is abbreviated as two dimensional or 2-DE. It is a type of gel electrophoresis (that separates molecules on the basis of their rate of movement through a gel). It is usually used to analyze proteins. Proteins are separated on the basis of two properties by mass and charge in two dimensions on 2D gels. Now a day's 2-DE is widely used for the separation of protein.
Methods of Gel Electrophoresis
IEF or Iso- Electric Focusing
In case of IEF (Iso-Electric Focusing) sample of proteins are separated on the basis of net charge. A strip is used which contain pH gradient from 0 to 14. The pH at which net charge of protein is zero is known as Iso-electric focusing or PI. Moreover, negatively charged molecules moves through the pH gradient towards the "positive" end while positively charged molecules migrate towards the "negative" end. Charge will be zero when it reaches to that pH. For example, pH of serum albumin is 4.8 (acidic protein). In IEF proteins are separated horizontally.
SDS-PAGE or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SDS-PAGE is a technique of electrophoresis (a molecule with a net charge will move in an electric field) in which protein molecules are separated on the basis of mass. In SDS-PAGE proteins are separated vertically.
Load IEF strip (pH gradient) on gel apparatus (SDS-PAGE), separated sample according to charge will be separated on gel. On IEF strip, separation starts horizontally on the basis of net charge while in case of SDS-PAGE (gel apparatus) separation of proteins start vertically on the basis of mass.
Limitations of 2-D Gel
1. Resolution Problem
We cannot achieve good resolution, but we can improve the resolution of 2-D gel through various methods.
• We can increase the size of the strip of IEF as well as increase the size of gel apparatus, that's how we can improve the resolution.
• We can use multiple strips on one gel, now a day's six strips per gel apparatus is used. This concept is called Zoom gel. In each gel we use narrow range of pH and we divide our sample on the strip.
• We have to prefractionate our sample because sample size will be reduced by prefraction and protein will be separated finely. In other words, proteins will be separated on the basis of density, through the principle of Centrifugation (divided into layers).
• We cannot reuse our gel.
2. Sensitivity Problem
Sensitivity means ability to detect protein. If we take an example of Yeast, in one cell,
• Protein A is 1, 00,000 (abundant)
• Protein B is 10,000 (less abundant)
• Protein C is 100 (rare/scarce)
i. Rare proteins will not be detected on the gel apparatus. Rare proteins act as transcription factor, we have to detect them.
ii. Abundant proteins dominate/obscure the less abundant proteins.
To avoid these two problems we will increase the resolution and prefractionate our sample as we have done this in resolution problem.
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