MAPK14 Antibody / Mitogen-activated protein kinase 14 / p38 alpha (FY12762)

Immunohistochemical staining of MAPK14 using anti- MAPK14 antibody. MAPK14 was detected in a paraffin-embedded section of human breast 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 ug/ml rabbit anti- MAPK14 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Western blot analysis of MAPK14 using anti- MAPK14 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Jurkat whole cell lysates, Lane 2: human SW620 whole cell lysates, Lane 3: human Hela whole cell lysates, Lane 4: human CACO-2 whole cell lysates, Lane 5: rat lung tissue lysates, Lane 6: rat heart tissue lysates, Lane 7: mouse lung tissue lysates, Lane 8: mouse heart 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- MAPK14 antibody at 0.5 ug/ml overnight at 4oC, 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 Substratewith Tanon 5200 system. A major band is detected at ~38 kDa, consistent with the active phosphorylated form of MAPK14 (predicted ~41 kDa), with a weaker lower band near 36 kDa corresponding to dephosphorylated or partially processed species.

Immunohistochemical staining of MAPK14 using anti- MAPK14 antibody. MAPK14 was detected in a paraffin-embedded section of mouse brain 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 ug/ml rabbit anti- MAPK14 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunohistochemical staining of MAPK14 using anti- MAPK14 antibody. MAPK14 was detected in a paraffin-embedded section of rat brain 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 ug/ml rabbit anti- MAPK14 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunoprecipitating MAPK14 in Jurkat whole cell lysate. Western blot analysis of MAPK14 using anti- MAPK14 antibody; Lane 1: Jurkat whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti- MAPK14 antibody in Jurkat whole cell lysate; Lane 3: anti- MAPK14 antibody (2ug) + Jurkat whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti- MAPK14 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A major band is detected at ~38 kDa, consistent with the active phosphorylated form of MAPK14 (predicted ~41 kDa), with a possible weaker lower band near 36 kDa corresponding to dephosphorylated or partially processed species.

Flow Cytometry analysis of CACO-2 cells using anti- MAPK14 antibody. Overlay histogram showing CACO-2 cells stained with (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- MAPK14 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.