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?

This data was developed using the same antibody clone in a different buffer formulation (ab184699).

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.

 

 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184699).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184699).

Clone EPR17526 (ab218017) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 + ERK2 antibody [EPR17526] (PE). Please refer to ab212153 for protocol details.

Overlay histogram showing A431 cells stained with ab212153 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 90% methanol at -20°C for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab212153, 1/2500 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184699).

Clone EPR17526 (ab218017) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 + ERK2 antibody [EPR17526] (Alexa Fluor® 647). Please refer to ab208881 for protocol details.

ab208881 staining ERK1 + ERK2 in A431 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab208881 at a 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

 

Clone EPR17526 (ab218017) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 + ERK2 antibody [EPR17526] (Alexa Fluor® 488). Please refer to ab208564 for protocol details.

ab208564 staining ERK1 + ERK2 in A431 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab208564 at a 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in A431 cells fixed with 4% formaldehyde (10 min).

 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184699).