POLR2H Antibody

Flow Cytometry analysis of U87 cells using anti-POLR2H antibody. Overlay histogram showing U87 cells (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-POLR2H Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

IF analysis of POLR2H using anti-POLR2H antibody and anti-Tubulin Alpha antibody. POLR2H was detected in immunocytochemical section of U2OS 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 µg/mL rabbit anti-POLR2H Antibody and mouse anti-Tubulin Alpha antibody overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight®488 Conjugated Goat Anti-Mouse IgG 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.

Western blot analysis of POLR2H using anti-POLR2H antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: 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-POLR2H antigen affinity purified polyclonal antibody 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 Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for POLR2H at approximately 17 kDa. The expected band size for POLR2H is at 17 kDa.