Anti-POLR2J Antibody Picoband®

Western blot analysis of POLR2J using anti-POLR2J antibody (A11442-1).
Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human MCF-7 whole cell lysates,
Lane 2: human U2OS whole cell lysates,
Lane 3: human PC-3 whole cell lysates,
Lane 4: human U251 whole cell lysates,
Lane 5: rat RH35 whole cell lysates,
Lane 6: rat C6 whole cell lysates,
Lane 7: mouse HEPA1-6 whole cell lysates,
Lane 8: mouse Neuro-2a 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-POLR2J antigen affinity purified polyclonal antibody (A11442-1) at 0.5 μg/mL overnight at 4°C, 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 is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for POLR2J at approximately 13 kDa. The expected band size for POLR2J is at 13 kDa.

IHC analysis of POLR2J using anti-POLR2J antibody (A11442-1).
POLR2J was detected in a paraffin-embedded section of human testis 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 μg/ml rabbit anti-POLR2J Antibody (A11442-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IF analysis of POLR2J using anti-POLR2J antibody (A11442-1) and anti-Alpha Tubulin antibody (M03989-3).
POLR2J was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-POLR2J Antibody (A11442-1) and mouse anti-Alpha Tubulin antibody (M03989-3) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and Fluoro488 Conjugated Goat Anti-Mouse IgG (BA1126) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunoprecipitating POLR2J in U251 whole cell lysate.
Western blot analysis of POLR2J using anti-POLR2J antibody (A11442-1).
Lane 1: U251 whole cell lysates (30ug),
Lane 2: Rabbit control IgG instead of anti-POLR2J antibody in U251 whole cell lysate,
Lane 3: anti-POLR2J antibody (2μg) + U251 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-POLR2J antigen affinity purified polyclonal antibody (A11442-1) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1197). A specific band was detected for POLR2J at approximately 13 kDa. The expected band size for POLR2J is at 13 kDa.