Protein extraction for two‐dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds
Top Cited Papers
- 16 July 2003
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 24 (14) , 2369-2375
- https://doi.org/10.1002/elps.200305500
Abstract
The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two‐dimensional electrophoresis (2‐DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water‐soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well‐resolved 2‐DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg⋅g−1 of aged leaf. We also evaluated the protocol by immunoblotting with anti‐tyrosinate α‐tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.Keywords
This publication has 20 references indexed in Scilit:
- Tubulin Post‐Translational ModificationsEuropean Journal of Biochemistry, 1997
- Two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimensin (IPG‐Dalt): The state of the art and the controversy of vertical versus horizontal systemsElectrophoresis, 1995
- Distribution of microtubules during the growth of tobacco pollen tubesBiology of the Cell, 1993
- Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast ExtractsPlant Physiology, 1992
- Preparation by two‐dimensional electrophoresis of proteins for antibody production: Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplastsElectrophoresis, 1988
- Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250Electrophoresis, 1988
- Technical improvements in two‐dimensional electrophoresis increase the level of genetic variation detected in wheat‐seedling proteinsElectrophoresis, 1986
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970