POLR1E Antibody / DNA-directed RNA polymerase I subunit E / PAF53 (FY12743)

Immunofluorescent staining of POLR1E using anti-POLR1E antibody (green) and anti-Beta Tubulin antibody (red). POLR1E was detected in an immunocytochemical section of U2OS 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-POLR1E 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 POLR1E using anti-POLR1E antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela 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-POLR1E 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. The expected band at ~50 kDa appears as a doublet, consistent with reported phosphorylation-dependent variants of POLR1E.

Immunoprecipitating POLR1E in RT4 whole cell lysate. Western blot analysis of POLR1E using anti-POLR1E antibody. Lane 1: RT4 whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-POLR1E antibody in RT4 whole cell lysate, Lane 3: anti-POLR1E antibody (2ug) + RT4 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-POLR1E 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. The expected band at ~50 kDa appears as a doublet in the input and immunoprecipitated samples, consistent with reported phosphorylation-dependent variants of POLR1E.