RANBP3 Antibody / Ran-binding protein 3 (FY13005)

Immunohistochemical staining of RANBP3 using anti-RANBP3 antibody. RANBP3 was detected in a paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-RANBP3 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Western blot analysis of RANBP3 using anti-RANBP3 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 1200V (Resolving gel) for 2-3 hours. Lane 1: human HepG2 whole cell lysates, Lane 2: human PC-3 whole cell lysates, Lane 3: human U251 whole cell lysates, Lane 4: human RT4 whole cell lysates, Lane 5: rat heart tissue lysates, Lane 6: rat C6 whole cell lysates, Lane 7: mouse heart tissue lysates, Lane 8: mouse NIH/3T3 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-RANBP3 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 an ECL Plus Western Blotting Substrate. A predominant band is detected at ~70 kDa, running above the ~60 kDa prediction, consistent with phosphorylation and the known slower migration of RANBP3. A weaker upper band at ~90 kDa is observed in some samples, consistent with SUMOylated/ubiquitinated RANBP3.

Immunofluorescent staining of RANBP3 using anti-RANBP3 antibody and anti-Tubulin Alpha antibody. RANBP3 was detected in immunocytochemical section of U2OS 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-RANBP3 antibody and mouse anti-Tubulin Alpha antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and Cy3 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of PC-3 cells using anti-RANBP3 antibody. Overlay histogram showing PC-3 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-RANBP3 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 without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.