Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides.
Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.
Nagai
Kouhei
K
Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Agency, Wakayama, Wakayama, Japan.
Yotsukura
Norishige
N
Ikegami
Haruka
H
Kimura
Hajime
H
Morimoto
Koichi
K
D020418:Algal Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000431:Ethanol; D044423:Kelp / Q000737:chemistry*; D019800:Phenol; D011134:Polysaccharides / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014783:Viscosity
D020418:Algal Proteins; D011134:Polysaccharides; D019800:Phenol; D012967:Sodium Dodecyl Sulfate; D000431:Ethanol