Monday, February 16, 2009

Understanding Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a method for the separation and identification of proteins in a sample by displacement in two-dimensions oriented at right angles to one another. This allows the sample to separate over a larger area, increasing the resolution of each component.

Two-dimensional gel electrophoresis is generally used as a component of proteomics and is the step used for the isolation of proteins for further characterization by mass spectroscopy. In the lab we use this technique for two main purposes. Firstly, for the large-scale identification of all proteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be more readily identified from mass spectrometry data. The second use of this technique is differential expression; this is when two or more samples are compared to find differences in their protein expression.

Two different protein-separating techniques are combined in sequence to achieve the goal of protein separation and identification-Iso electric Focusing (IEF) and SDS-PAGE. Isoelectric focusing (IEF) is used in the first-dimension. This separates proteins by their charge (pI) and SDS-PAGE (sodium dodecyle sulphate-polyacrylamide gel electrophoresis) is used in the second-dimension. This separates proteins by their size (molecular weight, MW). The procedure is known as ISO-OALT (iso for isoelectric focusing and dalt for molecular weight in dalton).

Isoelectric focusing (IEF). The side chains of amino acid residues of a protein contribute a net charge for protein molecules, which depend on the pH of the medium. In simple electrophoresis the mobility of the protein molecules is dependent on its charge that is controlled by the pH. There is a pH for every protein molecule at which the net charge of the protein becomes zero. This pH is known as isoelectric pH or isoelectric point (PI). At isoelectric pH the protein loses its mobility in the electric field. The technique of protein separation based on the property of isoelectric pH or PI is known as isoelectric focusing (IEF). A pH gradient is generated on an IEF gel and proteins are allowed to move in an electric field and that results in the separation of an individual protein species according to its isoelectric point.

Tags: Bio Technology, Bio Genetics, Proteins

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