Anti-p38 delta/MAPK13 + p38 alpha/MAPK14 antibody [M138] (ab31828)

IHC image of ab31828 staining p38 in normal human esophagus formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab31828, 1/50 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: p38 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab31828 observed at 40 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab31828 was shown to specifically react with p38 when p38 knockout samples were used. Wild-type and p38 knockout samples were subjected to SDS-PAGE. ab31828 and ab181602 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: p38 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab31828 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab31828 and a competitor's top cited rabbit polyclonal antibody.

Immunocytochemical labeling of activated p38 MAPK in pervanadate-treated mouse with ab31828. The cells were labeled with mouse monoclonal p38α MAPK and p38 MAPK antibodies, then the antibodies were detected using appropriate secondary antibodies conjugated to Cy3.

Overlay histogram showing A431 cells stained with ab31828 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab21828, 1:100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1:500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in A431 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Immunocytochemical analysis of mouse embryo fibroblast cells (NIH-3T3), labelling p38 with ab31828. Sample fixed in paraformaldehyde and blocked with 1% Donkey Serum + 1% BSA + 0.1% Triton X-100, in PBS for 30 minutes at 22°C. Incubated with ab31828 diluted 1/500 in 1% Donkey Serum + 1% BSA + 0.1% Triton X for 1 hour at 22°C. Secondary antibody was a Donkey anti-Mouse polyclonal conjugated to Alexa Fluor® 488, diluted 1/500. 

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