Recombinant Anti-ERK1 antibody [Y72] (ab32537)

Lanes 1- 2: Merged signal (red and green). Green - ab32537 observed at 43 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32537 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. ab32537 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.

 Overlay histogram showing HAP1 wildtype (green line) and HAP1-MAPK3 knockout cells (red line) stained with ab32537. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab32537, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. A Rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MAPK3 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. 

ab32537 staining ERK1 in wild-type HAP1 cells (top panel) and ERK1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 with ab32537 at 1/400 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labelling ERK1 with purified ab32537 at a dilution of 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Overlay histogram showing HeLa cells stained with ab32537 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween 20 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 (ab32537, 1/11312) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.01µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween 20 for 20 min used under the same conditions. 

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 - ab32537 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
ab32537 was shown to specifically react with ERK1 when ERK1 knockout samples were used.
Wild-type and ERK1 knockout samples were subjected to SDS-PAGE. ab32537 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) andGoat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

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 ab32537 and a competitor's top cited rabbit polyclonal antibody.

IHC image of ab32537 staining ERK1 in Human tonsil 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 ab32537, 2μg/ml 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 secondary only 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling ERK1 with unpurified ab32537.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling ERK1 with purified ab32537 at a dilution of 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 (1/1000) 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 (1/1000).

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

Unpurified ab32537 staining ERK1 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. ab150081, a goat anti rabbit Alexa Fluor^® 488 (1/200) was used as the secondary antibody. Counterstained with DAPI. Cytoplasmic and nuclear staining shown.

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Intracellular Flow Cytometry analysis ofJurkat cells labelling ERK1 with purified ab32537 at a dilution of 1/30 (red). Cells were fixed with 4% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

Unpurified ab32537 staining ERK1 (green) in HEK293 cells by intracellular flow cytometry. Cells were fixed with paraformaldehyde and permeabilized with 70% methanol. The sample was incubated with the primary antibody (1/40 in PBS + 0.2% BSA + 0.1% sodium azide) for 1 hour at 22°C. A phycoerythrin-conjugated goat anti-rabbit IgG (1/100) was used as the secondary antibody. Gating Strategy: Live Cells. Purple plot represents isotype control. 

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ab32537 (purified) at a dilution of 1/20 immunoprecipitating ERK1 in Jurkat whole cell lysate.

Lane 1 (input): Jurkat whole cell lysate (10µg)

Lane 2 (+): ab32537 + Jurkat whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32537 in Jurkat whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/10000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.