Evidence that rNanogP8 proteins can spontaneously form high M.W species.

<p>(<b>A</b>) rNanogP8 proteins from HPCa1 and MCF7, before and after purification, were used in WB using the R&D goat pAb. Note that prior to purification, the HPCa1 and MCF7 rNanogP8 proteins migrated at ∼42 kD and ∼37 kD, respectively, with a minor upper band detected for both proteins. After purification, the intensity of the upper bands (in the rectangle) became significantly stronger. (<b>B–C</b>) rNanogP8 proteins (from the indicated cell types) stored at −80°C for 8 days (B) or 5 weeks (C) were used in protein purification. Aliquots (20 µl) of 4 fractions (Frac.) for each sample, together with NTERA-2 non-induced (N) or induced (I) bacterial lysate (B) or LNCaP total bacterial lysate (Lys) or supernatant (Sup) (C), were used in WB with the eBioscience mAb. The arrows indicate the ∼42 kD rNanogP8 proteins and right-hand brackets indicate high M.W ladders. (<b>D</b>) The HPCa5 and HPCa1 rNanogP8 proteins stored for 2 years was utilized in WB with the Kamiya pAb and eBioscience mAb, respectively. (<b>E</b>) Coomassie brilliant blue R-250 staining of rNanogP8 protein made from HPCa5 cDNA in pET-28a. The arrows indicate the high levels of rNanogP8 proteins under the induced conditions. M, protein marker; N, non-induced; I, induced by IPTG. (<b>F</b>) Coomassie brilliant blue R-250 staining of HPCa5 rNanogP8 proteins from refolding dialysis experiment. The inclusion bodies of rNanogP8 protein were first dissolved in 7 M urea and then subjected to dialysis against decreasing concentrations of urea. N, non-induced; I, induced by IPTG; S, soluble portion; P, pellet (precipitated portion). Arrows indicate rNanogP8 proteins of different molecular weights. (<b>G</b>) IP analysis confirms that the refolded bands are rNanogP8. The dialysis samples containing 1.5 M or 0.6 M urea were subjected to IP with the SC pAb (H-155) or RbIgG (control) followed by WB analysis with the eBioscience mAb. Arrows indicate rNanogP8 proteins of different molecular weights.