The extraction of proteins from macroalgae has always been difficult, primarily because of their high internal levels of polysaccharides and polyphenols. Several methods for protein extraction have been developed recently that allow the production of high quality extracts for 1-D SDS gel electrophoresis or 2-D SDS-polyacrylamide gel electrophoresis for proteomic analysis. However, none of the existing methods are applicable to the study of the phosphorylation state of kinases involved in stress responses of macroalgae, primarily because trichloroacetic acid and phenol are used for protein precipitation. In this work, we describe a novel method for protein extraction from macroalgae based on the counterintuitive use of commercial RNA purification kits; this method maintains the phosphorylation state of the kinases intact, as demonstrated by either polyclonal or monoclonal antibodies against the phosphorylated forms of mitogen-activated protein kinases using Western blot methodology. This method may be appropriate for wider use in the analysis of phosphoproteomes.
©2012 by Walter de Gruyter Berlin Boston