Abstract
Two-dimensional gel electrophoresis (2-DGE) has become the central tool for proteome analysis and especially for the comparison of changes in protein expression levels arising from disease progression or therapeutic treatment modalities. Very often there are also changes involving post-translational modifications, such as phosphorylation or glycosylation. In order to study specific changes in protein post-translational modification, as well as overall changes in protein expression, the standard approach is to run two separate gels. One gel is used for the detection of a specific feature, like glycosylation (typically after Western blotting), and the second gel is used for total protein detection (usually employing silver stain or SYPRO® Ruby protein gel stain). This approach does, however, have some inherent problems and limitations associated with it. In order to map the glycoproteins, detected on an electroblot membrane, to the total protein profile from gels, the duplicate gels must run very reproducibly. Often, the overall dimensions of the gels change during fixation, staining, and transfer to the membrane, making it difficult to superimpose the gel profile directly with the blot profile. In addition, heavily glycosylated proteins usually are of high molecular weight and are difficult to transfer by electroblotting, thus leading to their loss from the analysis. Often, very hydrophilic glycoproteins bind poorly to transfer membranes as well, leading to their inefficient detection. Another major drawback to the procedure is that since numerous manipulations are involved in electroblotting, reliable quantitation becomes challenging.
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© 2003 Springer Science+Business Media New York
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Schulenberg, B., Patton, W.F. (2003). Multiplexed Proteomics. In: Conn, P.M. (eds) Handbook of Proteomic Methods. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59259-414-6_8
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DOI: https://doi.org/10.1007/978-1-59259-414-6_8
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61737-504-0
Online ISBN: 978-1-59259-414-6
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