Comparative fluorescence two-dimensional gel electrophoresis
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Comparative fluorescence two-dimensional gel electrophoresis (CoFGE) was developed to improve standardisation and reduce gel-to-gel variability in two-dimensional gel electrophoresis of proteins.
Overview[edit]
Gel electrophoresis is a standard method in the biochemical laboratory. In combination with isoelectric focussing, it is used to separate entire proteomes in two dimensions (2-DE). Comparative analysis of protein 2D-gel images is limited. Thus, difference gel electrophoresis was invented to overcome some of the limitations. This method is, however, not suitable for projects conducted over large periods of time or in different laboratories, because it requires the parallel handling of all replictate samples. CoFGE addresses this problem.[1] By introducing a sample-unrelated internal reference grid to the 2D-gel, it is possible to accommodate gel-to-gel variation and improve the overlay of protein spots and thus archiving of spot coordinates.
Technique[edit]
For CoFGE, a fluorescently labeled mixture of commercial proteins is co-separated with the analyte proteome in the second dimension. CoFGE was first developed for vertical 2-DE[2][3], but is easier to perform using horizontal gel electrophoresis.[4] The labeled mixture of reference proteins is introduced to the second dimension of the 2-DE process in a manner derived from 1-DE. To that end, wells for the standard mixture are created in equal distances on top of the gel close to the pI-strip holding the analyte proteome. In that way, during running of the gel, the reference proteins exhibit the same gel environment as the analyte and can subsequently be used to correct spot location by triangulation[1]. Since this procedure only corrects for variation in the y-dimension of the gel, an additional correction (x-dimension) is possible by using amphoteric dyes during isoelectric focussing.[5] CoFGE has been commercialized and gel analysis software has been adapted for the purpose (see manual[6] and updates in Delta 2D software; for review, see[1][7][8]). The general workflow has been published.[9] The CoFGE principle can also be applied for protein quantification, because the fluorescence intensity correlates with the protein concentration.[10][11] The method was awarded with the WWU Transfer Prize 2013/2014.[12]
References[edit]
- ↑ 1.0 1.1 1.2 König, Simone (1 January 2020). "Differential vs. comparative gel electrophoresis: New technology drives standardisation and quantification in protein two-dimensional gel electrophoresis". TrAC Trends in Analytical Chemistry. 122: 115731. doi:10.1016/j.trac.2019.115731. ISSN 0165-9936.
- ↑ Ackermann, Doreen; Wang, Weiqun; Streipert, Benjamin; Geib, Birgit; Grün, Lothar; König, Simone (May 2012). "Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid: Proteomics and 2DE". ELECTROPHORESIS. 33 (9–10): 1406–1410. doi:10.1002/elps.201200039.
- ↑ Koenig, Simone; ACKERMANN, Doreen; WEIQUN, Wang; GRUEN, Lothar (29 November 2012). "Improved 2d-gel electrophoresis". Google Patents.
- ↑ Hanneken, Marina; König, Simone (April 2014). "Horizontal comparative fluorescence two-dimensional gel electrophoresis for improved spot coordinate detection: Proteomics and 2DE". ELECTROPHORESIS. 35 (8): 1118–1121. doi:10.1002/elps.201300507.
- ↑ Hanneken, Marina; Šlais, Karel; König, Simone (1 September 2015). "pI-Control in comparative fluorescence gel electrophoresis (CoFGE) using amphoteric azo dyes". EuPA Open Proteomics. 8: 36–39. doi:10.1016/j.euprot.2015.03.003. ISSN 2212-9685.
- ↑ Nippes, Alina; Ackermann, Doreen; König, Simone (2018). "Analysis of CoFGE experiments with Delta2D". Mercator Journal of Biomolecular Analysis.
- ↑ Ohlendieck, Kay (2023). Methods in Molecular Biology: Difference Gel Electrophoresis (2 ed.). Humana Press. Search this book on
- ↑ König, Simone. "Comparative Fluorescence Gel Electrophoresis (CoFGE)". Decodon Academy.
- ↑ König, Simone; Ackermann, Doreen (15 September 2021). "Comparative two-dimensional fluorescence gel electrophoresis". ProtocolExchange. doi:10.21203/rs.3.pex-1612/v1.
- ↑ Parker, Ricardo (2019). Proteomics : advances in research and applications (1 ed.). Hauppauge: Nova Science Publishers. ISBN 978-1-53616-440-4. Search this book on
- ↑ Nippes, Alina; Ackermann, Doreen; König, Simone (2019). "Quantification with Comparative Fluorescence Gel Electrophoresis (CoFGE)". Mercator Journal of Biomolecular Analysis.
- ↑ "Transfer Prize 2013/2014". Videos WWU. AFO WWU.
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