Pull-down assay
In vitro pull-down analyses were performed using 50 μg of GST-Get3b and either 20 μg of Alb3C-His or 20 μg of Alb4C-His in a total volume of 120 μl of 1x PBS. Negative control reactions were conducted using GST instead of GST-Get3b or the His-tagged constructs alone. The samples were incubated for 10 min at room temperature. Subsequently, 100 μl of a 50% slurry of glutathione resin (equilibrated in 1x PBS) were added and incubated for 30 min at room temperature. The mixture was transferred to Wizard® Mini Columns (Promega) and centrifuged briefly. The column was washed with 5 ml of 1xPBS buffer using a syringe. After a brief centrifugation of the column the proteins were eluted with 30 μl of 10 mM reduced glutathione in 1xPBS buffer. The load as well as the eluted samples were analyzed by SDS-PAGE and proteins detected by Coomassie staining or Western blotting.