Anti-MEK1 antibody [Y77]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MEK1 antibody [Y77] (AB32576)

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling MEK1 with purified ab32576 at 1/50 dilution (6.6 μg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 antibody [Y77] (AB32576)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling MEK1 with purified ab32576 at 1/100 dilution (3.28 μg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 antibody [Y77] (AB32576)

ab32576 (unpurified) at a 1/250 dilution staining MEK1 in human urinary bladder carcinoma tissue by IHC-P.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MEK1 antibody [Y77] (AB32576)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling MEK1 with purified ab32576 at 1/500 dilution (5 μg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MEK1 antibody [Y77] (AB32576)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling MEK1 with purified ab32576 at 1/30 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IP

Unknown

Immunoprecipitation - Anti-MEK1 antibody [Y77] (AB32576)

ab32576 (purified) at 1/20 dilution (2ug) immunoprecipitating MEK1 in Jurkat whole cell lysate. Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10ug
Lane 2 (+) : ab32576 & Jurkat whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32576 in Jurkat whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-MEK1 antibody [Y77] (ab32576)

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

Western blot : Anti-MAP2K1 antibody [Y77] (ab32576) staining at 1/10000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32576 was shown to bind specifically to MAP2K1. A band was observed at 45 kDa in wild-type A549 cell lysates with no signal observed at this size in MAP2K1 knockout cell line. To generate this image, wild-type and MAP2K1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MAP2K1 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

MAP2K1 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 45 kDa

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• WB

Unknown

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

All lanes:

Western blot - Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution

All lanes:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate

Predicted band size: 43 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : MEK1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : A431 whole cell lysate (20 μg)
Lane 4 : HepG2 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32576 (unpurified) observed at 43 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32576 was shown to specifically react with MEK1 in wild-type HAP1 cells as signal was lost in MEK1 knockout cells. Wild-type and MEK1 knockout samples were subjected to SDS-PAGE. ab32576 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MEK1 antibody [Y77] (ab32576)

Predicted band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

Blocking and diluting buffer and concentration : 1% BSA/TBST. The identity of the higher MW bands between 100kDa and 200kDa are unknown. Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. In Western blot, anti-MEK1 antibody (ab32576) loading control staining at 1/1000 dilution. Exposure time : 26 seconds.

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 HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

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

Lane 3:

HeLa 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 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

Blocking and diluting buffer and concentration : 1% BSA/TBST. The identity of the higher MW bands between 100kDa and 200kDa are unknown. Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. In Western blot, anti-MEK1 antibody (ab32576) loading control staining at 1/1000 dilution. Exposure time : 15 seconds.

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 wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane), at 20 µg

Lane 2:

Wild-type HAP1 treated with 100ng/ml nocodazole for 24h whole cell lysate (untreated membrane) at 20 µg

Lane 3:

untreated MEK1 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

MEK1 knockout HAP1 treated with 100ng/ml nocodazole for 24h whole cell lysate at 20 µg

Lane 5:

Untreated wild-type HAP1 whole cell lysate (phosphatase treated membrane), at 20 µg

Lane 6:

Wild-type HAP1 treated with 100ng/ml nocodazole for 24h whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 7:

Untreated MEK1 knockout HAP1 whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 8:

MEK1 knockout HAP1 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 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-MEK1 antibody [Y77] (AB32576)

All lanes:

Western blot - Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates 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: 45 kDa

false