After extensive washing with NETN buffer, the beads were eluted with SDS sample buffer and subjected to western blot analysis.
After washing three times with lysis buffer, proteins on beads were eluted with SDS sample buffer.
For protein elution, beads were eluted with elution buffer (8 M urea, 100 mM NaPO4 (pH 8.0), 100 mM imidazole) for 5 min at room temperature.
Following centrifugation and several washes in lysis buffer, proteins recovered on beads were eluted with SDS sample buffer and separated by SDS-PAGE.
The beads were eluted with elution buffer (0.1 M glycine HCl, pH 2.0), and 2× loading buffer was added.
Beads were eluted in elution buffer (1% SDS, 0.1 M sodium bicarbonate in water) at 65°C twice, 10 min each.
After washing the beads five times with lysis buffer, proteins bound to beads were eluted by boiling in SDS-PAGE loading buffer.
After washing six times with lysis buffer, proteins bound to Sepharose beads were eluted by boiling in sample buffer, separated by 10% SDS-PAGE under non-reducing conditions, and transferred onto a nitrocellulose membrane (Trans-Blot transfer medium; Bio-Rad).
Beads were washed four times with RIPA buffer, and the proteins bound to the beads were eluted with loading buffer and subjected to SDS-PAGE, as described above.
CUL2/RBX1 (1.5 μg) was pre-incubated with 1.5 μg Flag-UBXN7 (approximately 1.5 times molar excess to CUL2) for 30 minutes, followed by one hour incubation with 10 μl anti-Flag beads in buffer D. The proteins bound to beads were eluted by boiling in Laemmli buffer.
