All lanes : Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse heart lysates
Lane 3 : Mouse kidney lysates
Lane 4 : Mouse spleen lysates
Lane 5 : Rat brain lysates
Lane 6 : Rat heart lysates
Lane 7 : Rat kidney lysates
Lane 8 : Rat spleen lysates
Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 44, 42 kDa why is the actual band size different from the predicted?

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAPK1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 43, 41 kDa
Observed band size: 44 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab184699 observed at 44 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab184699 Anti-ERK1 + ERK2 antibody [EPR17526] was shown to specifically react with ERK2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265052 (knockout cell lysate ab257525) was used. Wild-type and ERK2 knockout samples were subjected to SDS-PAGE. ab184699 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 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.

 

Ab184699 staining ERK1 + ERK2 in HeLa cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. A Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) was used as the secondary antibody.

See Abreview

Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution + Recombinant Human ERK1 protein (ab43623) (ab43623) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 44 kDa why is the actual band size different from the predicted?

Recombinant full length ERK1 protein (ab43623) contains aa1-379.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling ERK1 + ERK2 with ab184699 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing both nuclear and cytoplasmic 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:
-ve control 1: ab184699 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 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.

Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution + Recombinant Human ERK2 protein (ab43625) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

Recombinant full length ERK2 protein (ab43625) contains aa1-360.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/50000 dilution

Lane 1 : A431 (Human epidermoid carcinoma) whole cell lysates
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 4 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysates
Lane 5 : Human fetal brain lysates
Lane 6 : Human fetal heart lysates
Lane 7 : Human fetal kidney lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

Lane 1 : Human fetal brain lysates
Lane 2 : Human fetal heart lysates

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: 43, 41 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of A431 (Human epidermoid carcinoma) cells labeling ERK1 + ERK2 with ab184699 at 1/440 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

ERK1 + ERK2 were immunoprecipitated from 1mg of PC-12 (Rat adrenal gland pheochromocytoma) whole cell extract with ab184699 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab184699 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: PC-12 whole cell extract. Lane 2: PBS instead of PC-12 whole cell extract.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

44kDa band represents ERK1. 42kDa band represents ERK2.