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The aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus  Caulerpa  (the invasive  C. taxifolia  and  C. cylindracea  and the autochthonous  C. prolifera ). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for  C. prolifera  and  C. cylindracea , and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For  C. taxifolia , instead, none of the tested protocols produced satisfactory results and further studies will be required.

Comparative evaluation of multiple protein extraction procedures from three species of the genus Caulerpa