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.