Gray mullet, Mugil cephalus L., is a cosmopolitan fish species, widely distributed in coastal waters, lagoons, and estuaries between latitudes 42° N and 42° S. This mullet is an economically important species for both aquaculture and commercial fisheries around the world. A new single-step purification procedure was developed to purify gray mullet vitellogenin (VTG), from 17β-estradiol (E2)-treated gray mullet plasma. This method was performed by high performance liquid chromatography (HPLC) technique, using weak anion-exchange chromatography with a discontinuous gradient of NaCl (buffer B was 25 mM Tris-HCl, pH 8.5 + 0.416 M NaCl) with steps of 16.6 mM for 5.50 min from 0 to 0.416 M NaCl. SDS-PAGE electrophoresis revealed the presence of an intense protein band in E2-treated fish with a MW of 190 KDa. The 190 kDa is also the major protein band in the eluted fractions. Lower molecular weight proteins, corresponding to breakdown products of VTG, were also visible. However, by performing native-PAGE electrophoresis followed by Coomassie blue stain, two pure and non-degraded protein bands were detected. The two bands can be attributed to a monomeric and a dimeric form of VTG, as it has been already observed for VTG purified from other fish plasma samples. A polyclonal antibody against gray mullet VTG was raised in rabbits and found to be specific for gray mullet VTG through western blot analysis. Additionally, an enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of VTG in plasma samples of gray mullet.
VITELLOGENIN IN GRAY MULLET (MUGIL CEPHALUS): ONE-STEP PURIFICATION, CHARACTERIZATION, AND ELISA DEVELOPMENT
PUCCIARELLI, Stefania;MIANO, Antonino;PALERMO, Francesco Alessandro;COCCI, PAOLO;MICOZZI, DANIELA;MOSCONI, Gilberto
2015-01-01
Abstract
Gray mullet, Mugil cephalus L., is a cosmopolitan fish species, widely distributed in coastal waters, lagoons, and estuaries between latitudes 42° N and 42° S. This mullet is an economically important species for both aquaculture and commercial fisheries around the world. A new single-step purification procedure was developed to purify gray mullet vitellogenin (VTG), from 17β-estradiol (E2)-treated gray mullet plasma. This method was performed by high performance liquid chromatography (HPLC) technique, using weak anion-exchange chromatography with a discontinuous gradient of NaCl (buffer B was 25 mM Tris-HCl, pH 8.5 + 0.416 M NaCl) with steps of 16.6 mM for 5.50 min from 0 to 0.416 M NaCl. SDS-PAGE electrophoresis revealed the presence of an intense protein band in E2-treated fish with a MW of 190 KDa. The 190 kDa is also the major protein band in the eluted fractions. Lower molecular weight proteins, corresponding to breakdown products of VTG, were also visible. However, by performing native-PAGE electrophoresis followed by Coomassie blue stain, two pure and non-degraded protein bands were detected. The two bands can be attributed to a monomeric and a dimeric form of VTG, as it has been already observed for VTG purified from other fish plasma samples. A polyclonal antibody against gray mullet VTG was raised in rabbits and found to be specific for gray mullet VTG through western blot analysis. Additionally, an enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of VTG in plasma samples of gray mullet.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
