Ab81371

In total, 500 μL of Protein G Agarose beads (Roche) were used to capture 300 μL of SUMO-2/3 antibody (8A2, acquired from Abcam, ab81371; ∼5-10 μg/μL antibody). All washing and handling steps were followed by centrifugation of the beads at 500g for 3 min in a swing-out centrifuge with delayed deceleration, and careful aspiration of buffers. Beads were pre-washed 4 times with ice-cold PBS, after which the antibody was added in a 1.5 mL tube, with the tube filled completely with ice-cold PBS. Beads and antibody were incubated at 4°C on a rotating mixer for 1 h, and subsequently washed 3 times with ice-cold PBS. Crosslinking of the antibody to the beads was achieved by addition of 1 mL of 0.2 M sodium borate, pH 9.0, which was freshly supplemented with 20 mM dimethyl pimelimidate (DMP). Crosslinking was performed for 30 min at room temperature on a rotating mixer, after which the crosslinking step was repeated once. SUMO-IP beads where then washed twice with ice-cold PBS, twice with 0.1 M glycine pH 2.8, and three times with ice-cold PBS, after which they were stored until use at 4°C in PBS supplemented with 10 mM sodium azide.

Cells were seeded on glass coverslips in 24-well plates at ∼40,000 cells per well, and fixed after 24 h by incubation for 15 min in 3.7% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 2 mM MgCl2 pH 6.9) at 37 °C. Cells were washed twice with PBS, and permeabilized with 0.1% Triton X-100 for 10 min, washed with PBST and blocked using TNB (100 mM TRIS pH 7.5, 150 mM NaCl and 0.5% Blocking Reagent (Roche)) for 30 min. Cells were incubated with Mouse α SUMO-2/3 antibody (ab81371, Abcam, 1:500) in TNB for 1 h. Cells were washed five times with PBST, and indicated with secondary antibody (Goat α Mouse Alexa 488 (Invitrogen), 1:500) in TNB for 1 h. Subsequently, cells were washed five times with PBST and dehydrated using alcohol, prior to embedding them in Citifluor (Agar Scientific) containing 400 ng per μl DAPI (Sigma) and sealing the slides with nail varnish. Images were recorded on a Leica SP5 confocal microscope system using 488 and 561 nm lasers for excitation and a × 63 lens for magnification, and were analysed with Leica confocal software.