application example

Roseobacter capsulatus cells, pelleted by centrifugation and resuspended in an equial volume of TE buffer and supernatant sample* were separated on 10% SDS-PAGE and blotted 1h to nitrocellulose. The "+" indicated R. capsulatus SB1003 (GTA positive) and "-" indicated R. capsulatus A1 (GTA capsid protein negative). Blots were blocked in 5% skim milk in TBST followed by incubation with anti-GTA antibodies (AS08 365) at dilution 1: 1000 at 4°C over night. After washes blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Santa Cruz Biotechnology, Santa Cruz, CA) and specific bands were detected with SuperSignal West Femto (Pierce Biotechnology).Exposure time was 30 seconds with CCD camera.

* - supernatant sample was obtained in a following way:cells were removed by two rounds of centrifugation at 17,000 g for 2 min. with sub-sample of the supernatant removed to a new tube. Two volumes of the cells or final culture supernatant were mixed with 1 volume of 3X SDS-PAGE sample buuffer (NEB).