TWO‐DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF BROWN ALGAL PROTEIN EXTRACTS¹

Abstract

High-quality protein extracts are required for proteomic studies, a field that is poorly developed for marine macroalgae. A reliable phenol extraction protocol using Scytosiphon gracilis Kogame and Ectocarpus siliculosus (Dillwyn) Lyngb. (Phaeophyceae) as algal models resulted in high-quality protein extracts. The performance of the new protocol was tested against four methods available for vascular plants and a seaweed. The protocol, which includes an initial step to remove salts from the algal tissues, allowed the use of highly resolving two-dimensional gel electrophoresis (2-DE) protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms.

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

“…Proteins were extracted based on Contreras et al (2008). Cells were washed from the filter with 800 mL of extraction buffer (1.5% [w/v] polyvinylpyrrolidone, 0.7 M Suc, 0.1 M potassium chloride, 0.5 M Tris-HCl, pH 7.5, 250 mM EDTA and 0.5% CHAPS), and 8 mL of protease inhibitor cocktail for plant and tissue extracts (Sigma-Aldrich) was added.…”

Proteomics in Seaweeds: Ecological Interpretations

“…The work described by Yotsukura et al (2010) presents a similar protocol to the one described by Contreras et al (2008). Here, proteins are extracted from the brown alga Saccharina japonica (Chromista, Ochrophyta), important kelp described principally on the coastal areas of northern Japan.…”

“…In macro and microalgae the protein extraction protocol must be optimized, due to the high concentration of photosynthetic pigments that are known to interfere with the resolution of the 2-DE gels (e.g. Contreras et al, 2008;Wang et al, 2003;Wong et al, 2006). Particularly, in macroalgae protein extraction is difficult due to a low concentration and the co-extraction of contaminants such as anionic polysaccharides, polyphenols and salts, which are highly concentrated in the tissue (Chinnasamy & Rampitsch, 2006;Cremer & Van de Walle, 1985;Flengsrub & Kobro, 1989;Mechin et al, 2003).…”

“…In another important proteomic work with macroalgae developed by Kim et al (2008) and published contemporarily with the Contreras et al (2008) work, using as models the red algae Bostrychia radicans and B. moritziana (Plantae, Rhodophyta), used a lysis buffer comprised principally by urea and thiourea (detailed protocol in Appendix B). Although these species belong to the same group of red algae like G. changii, the simplicity of this method utilized in comparison with the phenol one (Wong et al, 2006) is due to the morphological characteristics of this species (see image in Appendix B).…”

“…Wang et al, 2003;Contreras et al, 2008) (detailed protocol in Appendix A). Proteins of Nannochloropsis oculata (Chromista, Ochrophyta) are obtained in very short time compared to the other microalgae protocols, however, not with the same quality as the more complex protocols.…”