Anti-MEK5/MAP2K5 Antibody Picoband®

Western blot analysis of MEK5/MAP2K5 using anti-MEK5/MAP2K5 antibody (A03980-3).
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 30ug of sample under reducing conditions.
Lane 1: human HEK293 whole cell lysates,
Lane 2: human A549 whole cell lysates,
Lane 3: human K562 whole cell lysates,
Lane 4: human A549 whole cell lysates,
Lane 5: human Hela whole cell lysates,
Lane 6: human PC-3 whole cell lysates,
Lane 7: human SW620 whole cell lysates,
Lane 8: monkey COS-7 whole cell lysates,
Lane 9: rat testis tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-MEK5/MAP2K5 antigen affinity purified polyclonal antibody (Catalog # A03980-3) 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 (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MEK5/MAP2K5 at approximately 50KD. The expected band size for MEK5/MAP2K5 is at 50KD.

IHC analysis of MEK5/MAP2K5 using anti-MEK5/MAP2K5 antibody (A03980-3).
MEK5/MAP2K5 was detected in paraffin-embedded section of human skeletal muscle tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-MEK5/MAP2K5 Antibody (A03980-3) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IF analysis of MEK5/MAP2K5 using anti-MEK5/MAP2K5 antibody (A03980-3).
MEK5/MAP2K5 was detected in immunocytochemical section of T47D 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 5μg/mL rabbit anti-MEK5/MAP2K5 Antibody (A03980-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of 293T cells using anti-MEK5/MAP2K5 antibody (A03980-3).
Overlay histogram showing 293T cells stained with A03980-3 (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-MEK5/MAP2K5 Antibody (A03980-3, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.