Recombinant Anti-ERK2 antibody [E460] (ab32081)

ab32081 staining ERK2 in wild-type HeLa cells (top panel) and ERK2 knockout HeLa cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab32081 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Lanes 1-2: Merged signal (red and green). Green - ab32081 observed at 41 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab32081 Anti-ERK2 antibody [E460] 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. ab32081 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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.

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.

ab32081 was shown to specifically react with ERK2 (MAPK1) in wild type HAP1 cells. No band was observed when ERK2 (MAPK1) knockout samples were used. Wild-type and ERK2 (MAPK1) knockout samples were subjected to SDS-PAGE. ab32081 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

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.

Lanes 1 - 2: Merged signal (red and green). Green - ab32081 observed at 44 kDa. Red signal from loading control - ab8226 observed at 42 kDa or ab8245, observed at 37 kDa. 

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 Fluorr^® 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. 

Immunohistochemical analysis of Human fallopian tissue epithelium, staining ERK2 with ab32081 at 1/50 dilution. Samples were incubated with primary antibody for 1 hour at room temperature. Staining was detected using DAB.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.