Abstract
Phenol extraction of proteins is an alternative method to classical TCA-acetone extraction. It allows efficient protein recovery and removes nonprotein components in the case of plant tissues rich in polysaccharides, lipids, and phenolic compounds. We present here a tried and tested protocol adapted for two dimensional electrophoresis (2-DE) and further proteomic studies. After phenol extraction, proteins are precipitated with ammonium acetate in methanol. The pelleted proteins are then resuspended in isoelectric focusing buffer, and the protein concentration is measured with a modified Bradford assay prior to electrophoresis.
The important points for successful use of this protocol are (1) keeping samples at very low temperature during the first step and (2) careful recovery of the phenolic phase after the centrifugations, which are major features of this protocol.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Michaud, D. and Asselin, A. (1995) Application to plant proteins of gel electrophoretic methods. J. Chromatogr. A 698, 263–279.
Damerval, C., Zivy, M., Granier, F., and de Vienne, D. (1988) Two-dimensional electrophoresis in plant biology. Adv. Electrophoresis 2, 263–340.
Hurkman, W. J. and Tanaka, C. K. (1986) Solubilisation of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806.
Mijnsbrugge, K. V., Meyermans, H., Van Montagu, M., Bauw, G., and Boerjan, W. (2000) Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins. Planta 210, 589–598.
Mihr, C. and Braun, H. P. (2003) Proteomics in plant biology, in Handbook of Proteomics (Conn, P., ed.), Humana Press, Totowa, NJ, pp. 409–416.
Carpentier, S. B., Witters, E., Laukens, K., Deckers, P., Swennen, R., and Panis, B. (2005) Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497–2507.
Wang, W., Scali, M., Vignani, R., et al. (2003) Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24, 2369–2375.
Saravanan, R. S. and Rose, J. K. C. (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4, 2522–2532.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Lowry, H., Rosebrough, J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
Ramagli, L. S. and Rodriguez, L. V. (1985) Quantification of microgram amounts of protein in two dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6, 559–563.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Faurobert, M., Pelpoir, E., Chaïb, J. (2007). Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues. In: Thiellement, H., Zivy, M., Damerval, C., Méchin, V. (eds) Plant Proteomics. Methods in Molecular Biology, vol 355. Humana Press. https://doi.org/10.1385/1-59745-227-0:9
Download citation
DOI: https://doi.org/10.1385/1-59745-227-0:9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-635-1
Online ISBN: 978-1-59745-227-4
eBook Packages: Springer Protocols