RTF2 Antibody / Replication termination factor 2 (FY12426)

Immunofluorescent staining of RTF2 using anti-RTF2 antibody (green) and anti-Beta Tubulin antibody (red). RTF2 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-RTF2 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and DyLight 594 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 RTF2 using anti-RTF2 antibody. Lane 1: human MCF-7 whole cell lysates, Lane 2: human Siha whole cell lysates, Lane 3: human Caco-2 whole cell lysates, Lane 4: human PC-3 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-RTF2 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. RTF2 (~34 kDa predicted) was detected primarily at ~37 kDa with a weaker ~35 kDa band, consistent with phosphorylation-dependent mobility shifts and minor processing events described in replication stress studies.

Flow Cytometry analysis of U87 cells using anti-RTF2 antibody. Overlay histogram showing U87 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-RTF2 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.

Immunoprecipitation of RTF2 in MCF-7 whole cell lysate. Western blot analysis of RTF2 using anti-RTF2 antibody; Lane 1: MCF-7 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-RTF2 antibody in MCF-7 whole cell lysate; Lane 3: anti-RTF2 antibody (2ug) + MCF-7 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-RTF2 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. RTF2 (~34 kDa predicted) was detected primarily at ~37 kDa with a weaker ~35 kDa band, consistent with phosphorylation-dependent mobility shifts and minor processing events described in replication stress studies.