Anti-Rsk 2/MAPKAP Kinase 1b/RPS6KA3 Picoband® Antibody

Western blot analysis of RPS6KA3 using anti-RPS6KA3 antibody (A02215-2).
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 CACO-2 whole cell lysates,
Lane 2: human SH-SY5Y whole cell lysates,
Lane 3: human 293T whole cell lysates,
Lane 4: human A549 whole cell lysates,
Lane 5: rat skeletal muscle tissue lysates,
Lane 6: rat thymus tissue lysates,
Lane 7: mouse skeletal muscle tissue lysates,
Lane 8: mouse thymus tissue 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-RPS6KA3 antigen affinity purified polyclonal antibody (Catalog # A02215-2) at 0.25 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 (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RPS6KA3 at approximately 84 kDa. The expected band size for RPS6KA3 is at 84 kDa.

Immunoprecipitating RPS6KA3 in A549 whole cell lysate.
Western blot analysis of RPS6KA3 using anti-RPS6KA3 antibody (A02215-2).
Lane 1: A549 whole cell lysates (30ug)
Lane 2: Rabbit control IgG instead of anti-RPS6KA3 antibody in A549 whole cell lysate.
Lane 3: anti-RPS6KA3 antibody (2μg) + A549 whole cell lysate (500μg)
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-RPS6KA3 antigen affinity purified polyclonal antibody (A02215-2) 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 # AR1196-200). A specific band was detected for RPS6KA3 at approximately 84 kDa. The expected band size for RPS6KA3 is at 84 kDa.

IF analysis of RPS6KA3 using anti-RPS6KA3 antibody (A02215-2) and anti-Beta Tubulin antibody (M01857-3).
RPS6KA3 was detected in immunocytochemical section of SiHa cell. 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-RPS6KA3 Antibody (A02215-2) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and DyLight®594 Conjugated Goat Anti-Mouse IgG (BA1141) 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.

Flow Cytometry analysis of 293T cells using anti-RPS6KA3 antibody (A02215-2).
Overlay histogram showing 293T cells stained with A02215-2 (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-RPS6KA3 Antibody (A02215-2, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.