Purification and characterization of 30 kDa proteins. DEAE cellulose ion exchange chromatography (A) followed by Sephadex G25 gel filtration chromatography (B). The protein content for exemplary fractions (A) 33-40, (B) 53-60, was visualized using 1D-PAGE. Fractions 38-51 showed two bands in the 30 kDa region (M - 30 kDa marker; ~30, 31 kDa), fractions 12-37 three bands (~ 29, 30, 31); otherwise the gels showed no protein signals. Gel filtration allowed isolation of protein in a single band (fraction 55-60). Bands from fractions 34 (A) and 60 (B) were subjected to LC-MS/MS analysis. The samples showing only three bands of the 30 kDa proteins were pooled (PUR) and stored at -80°C for further analysis. (C) Oil red O lipoprotein staining of fractions 38 and 37 separated by 12% SDS-PAGE. (D) Crude PVFB protein was compared to PUR protein by native 7% 1D-PAGE. The latter was not resolved and detected at ~130 kDa (no marker available). (E) Electron micrograph of PUR protein, magnification 50.000.
Pakkianathan et al. BMC Biochemistry 2012 13:5 doi:10.1186/1471-2091-13-5