Anti-MEK1 抗体 [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.

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.

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.

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.

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.

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). 

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.

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