Anti-MEK1 + MEK2 antibody [EPR16667]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

ab178876 staining MEK1 + MEK2 in the human cell line HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Immunohistochemical analysis of paraffin-embedded Human renal medulla tissue labeling MEK1 + MEK2 with ab178876 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Renal tubule epithelial cells show strong cytoplasmic staining with some additional nuclear staining. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

MEK1 + MEK2 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab178876 at 1/120 dilution. Western blot was performed from the immunoprecipitate using ab178876 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : HeLa whole cell extract. Lane 2 : PBS instead of HeLa whole cell extract.

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

All lanes:

Immunoprecipitation - Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876)

Predicted band size: 43 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling MEK1 + MEK2 with ab178876 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/200 dilution (green). Cytoplasmic staining on NIH/3T3 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab178876 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/200 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling MEK1 + MEK2 with ab178876 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Alveolar epithelial cells show strong cytoplasmic staining with some additional nuclear staining. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling MEK1 + MEK2 with ab178876 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Alveolar epithelial cells show strong cytoplasmic staining with some additional nuclear staining. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876) at 1/20000 dilution

Lane 1:

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

Lane 2:

293T (Human epithelial cells from embryonic kidney) whole cell lysates at 20 µg

Lane 3:

A549 (Human lung carcinoma) whole cell lysates at 20 µg

Lane 4:

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

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 44 kDa

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• WB

Lab

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Lanes 1 - 4 : Merged signal (red and green). Green - ab178876 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab178876 was shown to react with MEK2 in wild-type HEK-293T cells in western blot with loss of signal observed in MAP2K2 knockout sample. Wild-type HEK-293T and MAP2K2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab178876 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 20000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876) at 1/20000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

MAP2K2 knockout HEK293T cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876) at 1/50000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate 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: 43 kDa

Observed band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

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

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

In Western blot, anti-MEK1+MEK2 antibody (ab178876) loading control staining at 1/1000 dilution.

All lanes:

Western blot - Anti-MEK1 (phospho T286) antibody [EPR27226-8] (<a href='/en-us/products/primary-antibodies/mek1-phospho-t286-antibody-epr27226-8-ab307509'>ab307509</a>) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

NIH/3T3 treated with 100ng/ml nocodazole for 24h whole cell lysate (untreated membrane) at 20 µg

Lane 3:

NIH/3T3 treated with 100ng/ml nocodazole for 24h whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

true

Exposure time: 82s

• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (AB178876)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876) at 1/50000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Rat brain lysate at 10 µg

Lane 3:

Mouse heart lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 44 kDa

false