All lanes : Anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody [EPR18444] (ab214036) at 1/1000 dilution

Lane 1 : Untreated Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) treated with 200 ng/ml PMA for 30 minutes whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining increased after PMA treatment (200 ng/ml, 30min), and LP treatment decreased the PMA induced staining. For the “pan” antibody, the signal is unchanged after PMA treatment (200 ng/ml, 30min), and LP treatment. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. 

Nuclear and weak cytoplasmic staining on human glioma is observed [PMID:17487353].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

ERK1 (phospho T202) + ERK2 (phospho T185) was immunoprecipitated from 0.35 mg of Jurkat (Human T cell leukemia cell line from peripheral blood) treated with 200 ng/ml PMA for 30 minutes whole cell lysate with ab214036 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab214036 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1: Jurkat treated with 200 ng/ml PMA for 30 minutes whole cell lysate 10µg (Input).

Lane 2: ab214036 IP in Jurkat treated with 200 ng/ml PMA for 30 minutes whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab214036 in Jurkat treated with 200 ng/ml PMA for 30 minutes whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

Dot blot analysis of ERK1 (pT202) peptide (Lane 1), ERK1 (pT204) peptide (Lane 2), ERK1 (pT202 + pT204) peptide (Lane 3) and ERK1 non-phospho peptide (Lane 4) labelling ERK1 (pT202) with ab214036.

Exposure time: 3 minutes.

All lanes : Anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody [EPR18444] (ab214036) at 1/1000 dilution

Lane 1 : Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 50 ng/ml PDGF for 40 minutes whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on human normal breast tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling -ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining increased after PMA treatment (200 ng/ml, 30min), and LP treatment decreased the PMA induced staining.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab214036 at 1/100 dilution followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

ERK1 (phospho T202) + ERK2 (phospho T185) was immunoprecipitated from 0.35 mg of PC-12 (Rat adrenal gland pheochromocytoma cell line) treated with 200 ng/ml NGF for 4 days whole cell lysate with ab214036 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab214036 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1: PC-12 treated with 200 ng/ml NGF for 4 days whole cell lysate 10µg (Input).

Lane 2: ab214036 IP in PC-12 treated with 200 ng/ml NGF for 4 days whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab214036 in PC-12 treated with 200 ng/ml NGF for 4 days whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

All lanes : Anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody [EPR18444] (ab214036) at 1/1000 dilution

Lane 1 : Untreated PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) treated with 200 ng/ml NGF for 4 days whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 43, 41 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weak cytoplasmic staining on scattered cells of human placenta is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weak cytoplasmic staining on human breast tissue cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weak cytoplasmic staining on scattered cells of mouse kidney is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling ERK1 (phospho T202) + ERK2 (phospho T185) with ab214036 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weak cytoplasmic staining on scattered cells of rat spleen is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.