All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453) at 1/1000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Wild-type HEK-293T cell lysate
Lane 4 : MAPK14 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 41 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab182453 observed at 40 kDa. Red - loading control, ab130007 observed at 125 kDa.  

 ab182453 was shown to react with p38 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255406 (knockout cell lysate ab263787) was used. Wild-type and p38 knockout samples were subjected to SDS-PAGE. ab182453 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Predicted band size: 41 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab182453 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab182453 was shown to specifically react with p38 when p38 knockout samples were used. Wild-type and p38 knockout samples were subjected to SDS-PAGE. ab182453 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 3 : Neuro-2a (Mouse neuroblastoma cells) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 41 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling p38 with ab182453 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing cytoplasm and nucleus staining on Jurkat cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab182453 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

ab182453 staining p38 in the human cell line Jurkat (human acute T cell leukemia) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/180. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453)

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

Lysates/proteins at 20 µg per lane.

Predicted band size: 41 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab182453 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab182453 and a competitor's top cited rabbit polyclonal antibody.

All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 41 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-p38 alpha/MAPK14 antibody [EPR16878] (ab182453) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat heart lysate
Lane 5 : Rat kidney lysate
Lane 6 : Rat spleen lysate
Lane 7 : C6 (Rat glial tumor cells) whole cell lysate
Lane 8 : RAW 264.7(Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 9 : PC12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 10 : NIH 3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 41 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

5% NFDM/TBST: Blocking and diluting buffer.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling p38 with ab182453 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing cytoplasm and nucleus staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab182453 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunoprecipitation of p38 from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate achieved using ab182453 at 1/100 dilution.
Lane 1: Input: 10µg of Jurkat whole cell lysate.
Lane 2: Jurkat whole cell lysate following IP with ab182453.
Lane 3: negative control: IP using Rabbit monoclonal IgG (ab172730) instead of ab182453 in Jurkat whole cell lysate.
Western blot was performed using ab182453 at 1/1000 dilution.
An Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500 was used as secondary antibody.
Blocking and dilution buffer and concentration: 5% NFDM/TBST. 10 second exposure.