Figure 1. Western blot analysis of POLR2H using anti-POLR2H antibody (MBS1759193).
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 (#MBS1759193) at 0.5ug/mL overnight at 4 degree 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 (#MBS176460) 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.
Figure 2. IF analysis of POLR2H using anti-POLR2H antibody (MBS1759193) and anti-Tubulin Alpha antibody (M03989-3).
POLR2H was detected in immunocytochemical section of U2OS cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (#MBS176582) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5ug/mL rabbit anti-POLR2H Antibody (MBS1759193) and mouse anti-Tubulin Alpha antibody (M03989-3) overnight at 4 degree C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and DyLight488 Conjugated Goat Anti-Mouse IgG (BA1126) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37 degree C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 3. Flow Cytometry analysis of U87 cells using anti-POLR2H antibody (MBS1759193).
Overlay histogram showing U87 cells stained with MBS1759193 (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 (MBS1759193, 1ug/1x106 cells) for 30 min at 20 degree C. DyLight488 conjugated goat anti-rabbit IgG (MBS177405, 5-10ug/1x106 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1ug/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
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