RAI1 Antibody / Retinoic acid-induced protein 1 (FY12667)

Immunofluorescent staining of RAI1 using anti-RAI1 antibody (green) and anti-Beta Tubulin antibody (red). RAI1 was detected in an immunocytochemical section of cells. 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-RAI1 antibody and mouse anti-Beta Tubulin 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. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of RAI1 using anti-RAI1 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human RT4 whole cell lysates, Lane 3: human Hacat whole cell lysates, Lane 4: human MCF-7 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-RAI1 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 observed at ~250-260 kDa, which is widely reported for endogenous RAI1 despite the calculated ~203 kDa, consistent with known anomalous migration of this large nuclear regulator.

Flow Cytometry analysis of MCF-7 cells using anti-RAI1 antibody. Overlay histogram showing MCF-7 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-RAI1 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.