Anti-ERK2 antibody [E460]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ERK2 antibody [E460] (AB32081)

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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK2 antibody [E460] (AB32081)

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.

Image from Emmanuel C et al. PLoS One. 2011 Mar 15;6(3):e17617. doi: 10.1371/journal.pone.0017617.; Fig 2.; March 15 2011 PLoS ONE 6(3): e17617.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ERK2 antibody [E460] (AB32081)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK2 antibody [E460] (AB32081)

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.

• WB

Lab

Western blot - Anti-ERK2 antibody [E460] (AB32081)

ab32081 was shown to react with MAPK1 in wild-type HeLa cells in Western blot with loss of signal observed in MAPK1 knockout cell line ab265052. Wild-type HeLa and MAPK1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab32081 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-ERK2 antibody [E460] (ab32081) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 20 µg

Lane 2:

Western blot - Human MAPK1 (ERK2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mapk1-erk2-knockout-hela-cell-line-ab265052'>ab265052</a>) at 20 µg

Observed band size: 41 kDa

false

• WB

Lab

Western blot - Anti-ERK2 antibody [E460] (AB32081)

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.

All lanes:

Western blot - Anti-ERK2 antibody [E460] (ab32081)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

ERK2 knockout HAP1 cell lysate at 20 µg

Predicted band size: 41 kDa

false

• WB

Lab

Western blot - Anti-ERK2 antibody [E460] (AB32081)

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.

All lanes:

Western blot - Anti-ERK2 antibody [E460] (ab32081) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Lane 3:

46C (Mouse neural progenitor, selected for Sox1 expression cell line) Whole Cell Lysate at 20 µg

Lane 4:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 20 µg

Lane 5:

Recombinant Human ERK1 protein (<a href='/en-us/products/unavailable/recombinant-human-erk1-protein-ab43623-ab43623'>ab43623</a>) (<a href='/en-us/products/unavailable/recombinant-human-erk1-protein-ab43623-ab43623'>ab43623</a>) at 20 µg

Lane 6:

Western blot - Recombinant Human ERK2 protein (<a href='/en-us/products/proteins-peptides/recombinant-human-erk2-protein-ab43625'>ab43625</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-790-ab175781'>ab175781</a>) at 1/10000 dilution

Predicted band size: 41 kDa

Observed band size: 41 kDa

false

• WB

Unknown

Western blot - Anti-ERK2 antibody [E460] (AB32081)

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.

All lanes:

Western blot - Anti-ERK2 antibody [E460] (ab32081) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MAPK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MAPK1 (ERK2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mapk1-erk2-knockout-hela-cell-line-ab265052'>ab265052</a>)

Predicted band size: 41 kDa

Observed band size: 41 kDa

false

• WB

Lab

Western blot - Anti-ERK2 antibody [E460] (AB32081)

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.

All lanes:

Western blot - Anti-ERK2 antibody [E460] (ab32081)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

EKR2 knockout HAP1 cell lysate at 20 µg

Lane 3:

NIH3T3 cell lysate at 20 µg

Lane 4:

PC12 cell lysate at 20 µg

Predicted band size: 41 kDa

false