Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea)

Figure 2

Analysis and identification of adhesive protein in Cuvierian tubule extract from sea cucumber H. forskåli. Proteins were analyzed by 12% SDS PAGE and Western blotting. Gels were stained with Gel Code Blue Reagent. (A) Cuvierian tubule extract (4 M urea, 0.5 M Tris-HCl pH 7.5) purified with Ready Prep 2-D Clean up kit. (B) Western blot of Cuvierian tubule extract, incubated with antibody against precollagen D (1 : 1000 dilution) and developed with anti-rabbit IgG alkaline phosphatase. (C) Western blot detection of adhesive protein from H. forskåli, isolated by electroelution, using antibody against precollagen D of mussel M. galloprovincialis (1 : 1000 dilution) (positive control). (D) Western blot of Cuvierian tubule extract incubated with preimmune serum. (E) Western blot of Cuvierian tubule extract incubated with antibodies against isolated (electroeluted) adhesive protein from H. forskåli (dilution 1 : 1000); the serum was purified by treatment with Cuvierian tubule extract (dilution 1 : 10) at a ratio of 1 : 5. (F) Cuvierian tubule extract, separated on seminative gel. (G) Western blot of Cuvierian tubule extract, separated on seminative gel (dilution of antibody, 1 : 1000); the serum was purified by treatment with Cuvierian tubule extract (dilution 1 : 10) at a ratio of 1 : 10. M: Molecular mass markers. Arrowheads: adhesive protein (18 kDa) and dimer (36 kDa).