RING1 Antibody / Polycomb complex protein RING1 (FY13095)

Immunofluorescent staining of RING1 using anti-RING1 antibody (red) and anti-Beta Tubulin antibody (green). RING1 was detected in immunocytochemical section of cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-RING1 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG and FITC Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of RING1 using anti-RING1 antibody. Lane 1: human HEL whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human Jurkat whole cell lysates, Lane 4: rat PC-12 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RING1 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. Although RING1A is ~40-43 kDa by sequence, the antibody detects two major species: a ~50 kDa band and a ~38-40 kDa band. The upper band likely represents mono-ubiquitylated RING1A, while the lower band corresponds to the unmodified/shorter form.

Flow Cytometry analysis of human JK cells using anti-RING1 antibody. Overlay histogram showing JK cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-RING1 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample (Red line) was also used as a control.