All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilutionLane 1 : HEK293Lane 2 : JurkatLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

ICC/IF image of ab140601 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab140601 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilutionLane 1 : K6-linked-Ub2 recombinant proteinLane 2 : K27-linked-Ub2 recombinant proteinLane 3 : K29-linked-Ub2 recombinant proteinLane 4 : K11-linked-Ub2 recombinant proteinLane 5 : K48-linked-Ub2-7recombinant proteinLane 6 : K63-linked-Ub2-7 recombinant proteinLane 7 : K33-linked-Ub2 recombinant proteinLane 8 : monoubiquitin recombinant proteinLysates/proteins at 0.01 µg per lane.SecondaryHRP labelled goat anti-rabbit at 1/2000 dilution

Overlay histogram showing HeLa cells stained with ab140601 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab140601, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.