Immunoblots verify nef1 DNA also is expressed by r-GvpCNef1 and carried by recombinant gas vesicles. SDS-PAGE and Western blots demonstrate that Nef1 also is successfully expressed and can be detected in lysates and r-GV of SD109 transformed with pMS104D::nef1. The Coomassie Blue stained 12% SDS-PAGE gels shows resolution of proteins in gas vesicles or lysates from these transformed SD109 cultures (Panel A). Duplicates of these resolved proteins were electroblotted (Panel B) and the presence of the Nef1 peptide in chimeric r-GvpC, identified by both absorbed anti-Nef1 sera from mice immunized with r-GVNef1 and the monkey anti-SHIV antibody (plasma number R94085). Antibody binding shows recognition of Nef1 peptide within the recombinant GvpC protein. The mouse anti-Nef1 serum was used at a 1:250 dilution; the monkey anti-SHIV antibody was used at a 1:200 dilution. The standards (kDa) are shown on the left and the immuno detected band for recombinant Nef1 containing r-GvpC protein is identified on the right.
Sremac and Stuart BMC Biotechnology 2008 8:9 doi:10.1186/1472-6750-8-9