Ni2+-affinity purification of ASP-2. A detergent extract was prepared from a total of 50 × 106ASP-2-transfected cells as described in the methods, in the presence of protease inhibitors and loaded on an equilibrated Ni2+-charged agarose column, following dilution of the lysate to 0.5% (wt/vol) detergent and pure protein was eluted in fractions of 1 ml with 300 mM imidazole solution with 0.1% detergent. Eluates were loaded on a SDS-PAGE and proteins were detected by silver staining, (panel A) and anti-myc antibody developed by ECL (Panel B). Molecular weight standards are indicated in the first lane whereas the last lane shows a sample of elution buffer in loading buffer without protein. Typically, 20% yield was recovered following purification, amounting to approximately 40–60 μg of protein for a preparation from 50 × 106 cells. Solid arrows indicate the positions of the ASP-2 protein; the double solid arrow designates a pitative ASP-2 dimer.
Sidera et al. BMC Biochemistry 2002 3:25 doi:10.1186/1471-2091-3-25