Preparation of recombinant PA28γ and rabbit polyclonal antibodies to PA28γ. A) Coomassie stained SDS gel of purified recombinant PA28γ. Supernatant 1 and 2 (SN1 and SN2) contain the soluble E. coli proteins after the first two solubilization steps. Pelleted His-PA28γ precipitate was solved in 8 M urea. After dialysis insoluble His-PA28γ was removed by centrifugation (Pdia) and soluble recombinant protein (RP) was stored at 4°C. B) Immunoblot analysis with anti-PA28γ polyclonal antibody (crude serum, eluate of Protein A purification and eluate of PA28γ specific peptide column). Cellular extract of carcinoma cells (CE, 30 μg) and recombinant His-PA28γ protein (RP, 1 μg) were separated by SDS-PAGE, blotted and incubated with rabbit anti-PA28γ antibody purifications.