Figure 7.
Streptavidin purification of Al-HNE adducted proteins conjugated with biotin. Cellular lysates (Lane 1) containing Al-HNE adducted proteins conjugated with biotin (2 mg) were incubated with 1 ml of SA beads (1:1 in ammonium bicarbonate) for 2 h. After 2 h the beads were recovered by centrifugation and the breakthrough was collected (Lane 2). Successive washes include 1% SDS (Lane 3), 4 M urea (Lane 4), and 1 M NaCl (Lane 5). Elution of biotin was done with 70% CH3CN, 5% formic acid (Lane 6). Biotin tagged proteins were detected with HRP-SA and 2.5 μg of protein was loaded per lane. Panel A shows control (DMSO) and 5 μM Al-HNE and panel B shows 10 and 50 μM Al-HNE. S=biotin labeled standard curve.