Protein extraction for two‐dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds

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 alpha-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.

Method for obtaining high-resolution proteomic analysis from pericarps of guarana

“…Similar difficulties were reported by Wang et al (2003), who observed that when proteins of the olive leaf were extracted by direct homogenization in aqueous buffer and precipitated in organic solvent, polyphenols and other contaminants were adsorbed to the proteins, which caused the pellet to darken due to oxidation. This contamination hampers subsequent dissolution, making the protein extract unsuitable for 2-dimensional electrophoresis (2-DE) analysis.…”

“…A very efficient method involves the dissolution of proteins in phenol and precipitation in ammonium acetate in methanol (Hurkman and Tanaka, 1986). This method was successfully used for coffee (Franco et al, 2009), olive leaves (Wang et al, 2003), and other recalcitrant tissues such as those from tomato, avocado, and orange peel (Saravanan and Rose, 2004), as well as banana, apple, and potato (Carpentier et al, 2005). Despite the description of various biological properties of guarana and significant amounts of information that were obtained from its transcriptome, proteomic profiles from the different fruit tissues in the various developmental phases have not been reported.…”

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater

“…This method has the side effect (often in combination with SDS) to lyse efficiently bacteria for subsequent extraction of protein (Guerlava et al, 1998). The dark-brown color of NaOH extracts from untreated compost soil indicated that besides intracellular proteins of soil microorganisms, high amounts of humic organic matter were eluted, which we attempted to remove by a water and liquid phenol two phase extraction that had been used to purify proteins from lysates of olive leafs (Wang et al, 2003) and fungal spores (Benndorf, unpublished results). Phenol is also used to remove proteins in DNA and RNA purification protocols (Kirby, 1956), because its gentle hydrophobicity makes it a better solvent for proteins than water.…”

Proteomics Analysis of Drought Stress-Responsive Proteins in Hippophae rhamnoides L.

“…TCA precipitation in conjunction with phenol extraction, as described by Wang et al (2003), was used to extract proteins from fresh young leaves. The leaves were ground in liquid nitrogen using a mortar and pestle, the powder tissue was placed in 10-ml tubes (0.4 g tissue powder per 10-ml microtubes) and then resupended with 5 ml of −20°C acetone.…”