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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human ERK1 aa 1-100 (N terminal).
Database link: P27361
A trial size is available to purchase for this antibody.
Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab32537 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000. Detects a band of approximately 44 kDa (predicted molecular weight: 43 kDa).|
|IP||1/20 - 1/40.|
|Flow Cyt||1/30 - 1/40.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
|IHC-P||Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).
This product gave a positive signal in HAP1 cells fixed with 4% formaldehyde (10 min) and 100% methanol (5 min).
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) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
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).
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab32537 overnight at 4°C. Antibody binding was detected using a goat anti-rabbit Alexa Fluor® 790) ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
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.
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.
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).
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.
Flow Cytometry analysis of Jurkat 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.
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 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. 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.
Unpurified ab32537 staining ERK1 (green) in HEK293 cells by 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.
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 as the secondary antibody (1/10000).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.