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Improved protein solubility in two‐dimensional electrophoresis using tributyl phosphine as reducing agent
Abstract
In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first‐dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first‐dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second‐dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol‐specific alkylating reagents.
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Cited by
The current state of two-dimensional electrophoresis with immobilized pH gradients
Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., & Weiss, W. (2000) · Electrophoresis · doi:10.1002/(sici)1522-2683(20000401)21:6<1037::aid-elps1037>3.0.co;2-v · Cited by 1,515
Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J., Hochstrasser, D. F., Williams, K. L., & Gooley, A. A. (1998) · ELECTROPHORESIS · doi:10.1002/elps.1150190539 · Cited by 489
Self-citationAnalysis of the Arabidopsis Mitochondrial Proteome
Millar, A. H., Sweetlove, L. J., Giegé, P., & Leaver, C. J. (2001) · Plant Physiology · doi:10.1104/pp.010387 · Cited by 447
Peltier, J.-B., Friso, G., Kalume, D. E., Roepstorff, P., Nilsson, F., Adamska, I., & van Wijka, K. J. (2000) · The Plant Cell · doi:10.1105/tpc.12.3.319 · Cited by 355
Perspectives for mass spectrometry and functional proteomics
Godovac-Zimmermann, J., & Brown, L. R. (2001) · Mass Spectrometry Reviews · doi:10.1002/1098-2787(2001)20:1<1::aid-mas1001>3.0.co;2-j · Cited by 264
Metadata
- Authors
- Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., & Williams, K. L.
- Venue
- ELECTROPHORESIS
- Publisher
- Wiley
- Published
- 1998-05
- Volume
- 19
- Issue
- 5
- Pages
- 845-851
- ISSN
- 0173-0835 (ISSN-L)
- 1522-2683
- OpenAlex
- W2090014602
- Cited by
- 240
- References
- 0
- FWCI
- 10.38
- Type
- journal article
- Language
- en
- Open access
- closed
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- Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., & Williams, K. L. (1998). Improved protein solubility in two‐dimensional electrophoresis using tributyl phosphine as reducing agent. ELECTROPHORESIS. https://doi.org/10.1002/elps.1150190540
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