application example
STD: Protein standard (molecular weights of the proteins are indicated on the left) (1) plasma from male sole (load 0.11 ul of plasma),(2) plasma from vitellogenic female sole (load 0.11 ul of plasma), (3) plasma from estradiol-treated male sole (load 0.08 ul of plasma,(4) VTG precipitate (load 0.83 ul of precipitate),(5) purified VTG (load 2 ul of purified preparation, corresponding to around 0.14 ug and spawning performance in cultured VTG) were separated on SDS-PAGE 7.5% resolving gel, 4% stacking gel. Samples were denatured in SDS and b- mercaptoethanol and treated 4 min 95 oC before loading. Following gel electrophoresis proteinswere transfered to PVDF Membrane (Inmobilon-P, Millipore) for 2 h. Blots were blocked in TBST containing 2% non-fat dry milk. Blots were incubated in primary antibody at a dilution 1: 40 000, followed by incubation with secondary antibodies (GAR-HRP, BioRad, dilution 1:2,000) and chemiluminiscence development (Luminol Reagent, Santa Cruz Biotechnology).