RBM12B Antibody / RNA-binding protein 12B (FY12589)

Immunofluorescent staining of RBM12B using anti-RBM12B antibody (red) and anti-Beta Tubulin antibody (green). RBM12B was detected in immunocytochemical section of HELA 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-RBM12B 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 RBM12B using anti-RBM12B antibody. Lane 1: human whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human Jurkat 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-RBM12B 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. A prominent band is detected at ~130 kDa, slightly higher than the calculated ~118 kDa. The slower migration is consistent with reported observations for RBM12B and with phosphorylation- or low-complexity-region-related migration behavior typical of RNA-binding proteins.

Flow Cytometry analysis of cells using anti-RBM12B antibody. Overlay histogram showing 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-RBM12B 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.

Flow Cytometry analysis of JK cells using anti-RBM12B 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-RBM12B 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.

Immunoprecipitating (IP) RBM12B in whole cell lysate. Western blot analysis of RBM12B using anti-RBM12B antibody; Lane 1: whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-RBM12B antibody in whole cell lysate; Lane 3: anti-RBM12B antibody (2ug) + whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-RBM12B antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A specific band was detected for RBM12B at approximately 130 kDa. The expected molecular weight of RBM12B is at 118 kDa.