All lanes : Anti-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : MAPK3 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

Lanes 1- 2: Merged signal (red and green). Green - ab109282 observed at 43 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109282 was shown to react with ERK1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266519 (knockout cell lysate ab257099) was used. Wild-type HEK-293T and MAPK3 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109282 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling ERK1 with purified ab109282 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of Jurkat (human acute T cell leukemia) cells labelling ERK1 with purified ab109282 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling ERK1 with purified ab109282 at 1/30 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: ERK1 knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target - ab109282 observed at 42 kDa
Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal
ab109282 was shown to specifically react with ERK1 when ERK1 knockout samples were used.
Wild-type and ERK1 knockout samples were subjected to SDS-PAGE. ab109282 and ab8226 (loading control to beta actin) were both diluted 1/1000 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 1/10000 dilution for 1 h at room temperature before imaging.

Predicted band size : 43 kDa

Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: ERK1 knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target 
Lanes 3 and 4: Red signal from loading control 
Lanes 5 and 6: Merged (red and green) signal

Red - loading control, ab8226 observed at 42 kDa or ab8245, observed at 37 kDa

This western blot image is a comparison between ab109282 and a competitor's top cited rabbit polyclonal antibody.

All lanes : Anti-ERK1 antibody [EP4967] (ab109282) at 1/10000 dilution (purified)

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 44 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-ERK1 antibody [EP4967] (ab109282) at 1/10000 dilution (purified)

Lane 1 : Jurkat cell lysate
Lane 2 : A375 cell lysate
Lane 3 : HUVEC cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 44 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution (unpurified)

Lane 1 : Jurkat cell lysate
Lane 2 : A375 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HUVEC cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 44 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colonic adenocarcinoma tissue labelling ERK1 with unpurified ab109282 at 1/100 dilution.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.