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2D Poly-acryl Amide Gel Electrophoresis (2D-PAGE) is a biochemical tool for separating proteins based on isoelectric point as well as on charge and molecular weight of those proteins. It has great importance in the biochemical field and it can also be used to diagnose molecular diseases. Because molecular diseases are caused either due to the production of certain proteins or absence of certain proteins.
2 Dimensional Poly-Acryl Amide Gel Electrophoresis (2D-PAGE) is a type of poly-Acryl amide gel electrophoresis in which proteins are run in a gel strip first to separate based on the isoelectric point. For which gel strip is maintained in a gradient of increasing pH and, therefore, proteins are first separated based on the isoelectric point and lie in a particular pH zone. After isoelectric point separation, that gel strip is placed in the normal SDS-PAGE and run to separate them further based on molecular weight.
Molecular Disease Diagnosis
2 dimensional Poly-Acryl Amide Gel Electrophoresis has great importance in the detection of changes in gene expression between two different biological states, as for example comparison of normal and diseased tissue.
2D PAGE patterns of an extract from diseased tissue such as liver tumor can be compared with the 2D PAGE pattern of extract from normal liver tissue to see whether there are any differences in these two patterns. If a protein present (or is absent) only in the liver tumor, can lead us to an identification of that protein and gene responsible for the production of that protein.
Thus, it will be easy to understand why this gene is expressed (or not) in the diseased state. In this way, 2D PAGE can be utilized to understand the molecular basis of disease.
Up to 5000 protein spots can be identified and visualized in a large format of a 2D gel. Thus, with a 2D PAGE, we can follow changes in the expression of a significant proportion of a protein in a cell or tissue. However, it is necessary to produce a 2D map of the proteins expressed by an organism, tissue or cell under normal condition before making any comparison with a diseased one. Then 2D reference map and database can be used to compare similar identification between a normal or diseased or treated organism, tissue or cells. Using 2D PAGE we can analyze
- Effect of drug treatment or toxin on cell
- Effect of changing protein component of the cell at the different stage of tissue differentiation and development.
- Response to extracellular stimuli such as hormones or cytokines comparison of pathogenic and non-pathogenic bacterial strain.
Analyzing 2D-PAGE patterns with different proteomic databases
A research group studying the toxic effect of drugs on the liver can compare the 2D gel pattern from their damaged liver with normal liver 2D reference map, thus identifying protein changes that occur as a result of drug treatment. Now a day there is a range of commercial 2D gel analysis software available for personal computer workstations, which can be used to get qualitatively and quantitative information from the gel pattern and this software can also be used to compare the pattern of two different 2D gel.
This allows the construction of a range of databases of quantitative protein expression in a range of tissue and cell types. An extensive series of 2D database SWISS-2D-PAGE (maintained by the Geneva University Hospital) is accessible via www. This facility allows an individual laboratory to compare their own 2D-protein database with that in another laboratory using software packages like Flicker program available on http://open2dprot.sourceforge.net/Flicker/.
This program superimposes the two 2D patterns to be compared and then displays one pattern. Spots that appear in both gel patterns will be seen as fixed spot, but a spot that appears on one gel and not on the other gel will be seen to be flashing (flickering). Once the protein spot is identified, it is necessary to identify a specific protein, for which peptide mass fingerprinting is done. The spot of interest is cut out of the gel and incubated in a solution of proteolytic enzyme trypsin, which cleaves the protein C-terminal to each of the arginine and the lysine residue.
Peptide fractions are collected and then analyzed by MALDI-MS to determine an accurate mass of each peptide in that sample. Now using web-based programs like Mascot or protein Prospector, these peptide-mass-fingerprint are compared with the databases of trypsin peptide mass fingerprints generated from sequences of known proteins. Proteins can then be identified if any match is found with the fingerprint from the database.
Sometimes, results from peptide mass fingerprinting can be ambiguous. In such case partial amino acid sequence database from one of the peptide is required, which is done by the Tandem Mass Spectrometry. In Tandem Mass Spectrometry, one of the peptides separated from mass fingerprinting is further fragmented and from these fragmentation patterns, sequence data are deduced. Thus, obtained partial sequence data is used to search protein sequence databases for sequence identity.