Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human ERK2 aa 300 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: P28482
This product is a recombinant rabbit monoclonal antibody.
A trial size is available to purchase for this antibody.
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.
Our Abpromise guarantee covers the use of ab32081 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|WB||1/1000 - 1/2000. Detects a band of approximately 42 kDa (predicted molecular weight: 41 kDa).
Can be blocked with ab205612
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: ERK2 knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target - ab32081 observed at 44 kDa
Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal
ab32081 was shown to specifically react with ERK2 when ERK2 knockout samples were used. Wild-type and ERK2 knockout samples were subjected to SDS-PAGE. ab32081 and ab8226 (loading control to beta actin) were both diluted at 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 1/10 000 dilution for 1 h at room temperature before imaging.
IHC image of ab32081 staining ERK2 in rat pancreas 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 ab32081, 5μg/ml, 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.
Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ERK2 with ab32081 at 1/400. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: ERK2 knockout HAP1 cell lysate (20 µg)
This western blot image is a comparison between ab32081 and a competitor's top cited rabbit polyclonal antibody.
Overlay histogram showing HeLa cells stained with ab32081 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab32081, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor® 488 (IgG; H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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 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 ab32081 overnight at 4°C. Antibody binding was detected using ab175781 (goat anti-rabbit Alexa Fluor 790) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.