Anti-ERK1 + ERK2 抗体 [EPR17526]

• ICC

Lab

Immunocytochemistry - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

ab288063 staining ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) in untreated, PMA treated and PMA + LP treated HeLa cells. The cells were fixed with 100% Methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab288063 at 5µg/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 magenta). 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.

The image shows increased nuclear staining after 24hr serum starvation followed by treatment with PMA (200nM, 15min) of HeLa cells. The Lambda Phoshatase treatment then removes all staining of antibody with no phospho ERK1/2 remaining.

ab184699 was used as a Pan ERK1 + ERK2 control for ab288063. The results showed nuclear staining on untreated, with no increase after PMA treatment and no reduction after PMA + LP treated in HeLa cells.

• ICC/IF

AbReview59872****

Immunocytochemistry/ Immunofluorescence - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

ab184699 staining ERK1 + ERK2 in HeLa cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. A Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Kirk Mcmanus.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

Intracellular flow cytometric analysis of A431 (Human epidermoid carcinoma) cells labeling ERK1 + ERK2 with ab184699 at 1/440 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling ERK1 + ERK2 with ab184699 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing both nuclear and cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab184699 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• ICC

Lab

Immunocytochemistry - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

ab288063 staining ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) in untreated, PMA treated and PMA + LP treated NIH/3T3 cells. The cells were fixed with 100% Methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab288063 at 5µg/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 magenta). 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.

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

The image shows increased nuclear staining after 24hr serum starvation followed by treatment with PMA (200nM, 15min) of NIH/3T3 cells. The Lambda Phoshatase treatment then removes all staining of antibody with no phospho ERK1/2 remaining.

ab184699 was used as a Pan ERK1 + ERK2 control for ab288063. The results showed nuclear staining on untreated, with no increase after PMA treatment and no reduction after PMA + LP treated in NIH/3T3 cells.

• IP

Supplier Data

Immunoprecipitation - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

ERK1 + ERK2 were immunoprecipitated from 1mg of PC-12 (Rat adrenal gland pheochromocytoma) whole cell extract with ab184699 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab184699 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : PC-12 whole cell extract. Lane 2 : PBS instead of PC-12 whole cell extract.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

44kDa band represents ERK1. 42kDa band represents ERK2.

All lanes:

Immunoprecipitation - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699)

Predicted band size: 41 kDa

false

• WB

Supplier Data

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

Lane 1:

Human fetal brain lysates at 10 µg

Lane 2:

Human fetal heart lysates at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa,44 kDa

false

• WB

Lab

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

Lanes 1-2 : Merged signal (red and green). Green - ab184699 observed at 44 kDa. Red - loading control ab8245 observed at 37 kDa.

ab184699 Anti-ERK1 + ERK2 antibody [EPR17526] 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. ab184699 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 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-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 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='/products/cell-lines/human-mapk1-erk2-knockout-hela-cell-line-ab265052'>ab265052</a>)

Predicted band size: 41 kDa

Observed band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/50000 dilution

Lane 1:

A431 (Human epidermoid carcinoma) whole cell lysates at 20 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

Lane 3:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 20 µg

Lane 4:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysates at 20 µg

Lane 5:

Human fetal brain lysates at 20 µg

Lane 6:

Human fetal heart lysates at 20 µg

Lane 7:

Human fetal kidney lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa,44 kDa

false

• WB

Lab

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

ab288063 was shown to specifically react with ERK1/2 (pT202+ Y204). The signal was significantly reduced after treat with 10 uM U0126.
Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% BSA in TBS-0.1 % Tween® 20 (TBS-T).
ab288063, ab184699 and Anti-Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1/1000, 1/10000 and 1/5000 dilutions, respectively.
Blots were developed with Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ERK1 (phospho T202+Y204) + ERK2 (phospho T185+Y187) antibody [EPR25015-F2] (<a href='/products/primary-antibodies/erk1-phospho-t202y204-erk2-phospho-t185y187-antibody-epr25015-f2-ab288063'>ab288063</a>) at 1/1000 dilution

Lane 1:

HeLa cells, serum-starved overnight, treated with U0126 (10 uM, 60 min) at 20 µg

Lane 2:

HeLa cells, serum-starved overnight, treated with TPA (200 nM, 15 min) at 20 µg

Lane 3:

NIH3T3 cells, serum-starved overnight, treated with U0126 (10 uM, 60 min) at 20 µg

Lane 4:

NIH3T3 cells, serum-starved overnight, treated with TPA (200 nM, 15 min) at 20 µg

Lane 5:

A10 cells, serum-starved overnight, treated with U0126 (10 uM, 60 min) at 20 µg

Lane 6:

A10 cells, serum-starved overnight, treated with TPA (200 nM, 15 min) at 20 µg

Secondary

Lanes 1 - 6:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 6:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 42 kDa,44 kDa,50 kDa

false

• WB

Supplier Data

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

44kDa band represents ERK1. 42kDa band represents ERK2.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

Lane 1:

Mouse brain lysates at 10 µg

Lane 2:

Mouse heart lysates at 10 µg

Lane 3:

Mouse kidney lysates at 10 µg

Lane 4:

Mouse spleen lysates at 10 µg

Lane 5:

Rat brain lysates at 10 µg

Lane 6:

Rat heart lysates at 10 µg

Lane 7:

Rat kidney lysates at 10 µg

Lane 8:

Rat spleen lysates at 10 µg

Lane 9:

C6 (Rat glial tumor cells) whole cell lysates at 10 µg

Lane 10:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates at 10 µg

Lane 11:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 10 µg

Lane 12:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa,44 kDa

false

• WB

Supplier Data

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

Recombinant full length ERK1 protein (ab43623) contains aa1-379.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

All lanes:

Recombinant Human ERK1 protein (<a href='/products/unavailable/recombinant-human-erk1-protein-ab43623-ab43623'>ab43623</a>) (<a href='/products/unavailable/recombinant-human-erk1-protein-ab43623-ab43623'>ab43623</a>) at 0.01 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 41 kDa

Observed band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (AB184699)

Recombinant full length ERK2 protein (ab43625) contains aa1-360.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution

All lanes:

Western blot - Recombinant Human ERK2 protein (<a href='/products/proteins-peptides/recombinant-human-erk2-protein-ab43625'>ab43625</a>) at 0.01 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 41 kDa,52 kDa

Observed band size: 42 kDa

false