Anti-MEK1 antibody [E342]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MEK1 antibody [E342] (AB32091)

Overlay histogram showing HeLa cells stained with ab32091 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32091, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 antibody [E342] (AB32091)

ab32091, at a 1/100 dilution, staining MEK1 in paraffin embedded human cervical carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MEK1 antibody [E342] (AB32091)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling MEK1 with purified ab32091 at dilution of 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control.

• WB

Lab

Western blot - Anti-MEK1 antibody [E342] (AB32091)

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 - ab32091 observed at 43 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32091 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. ab32091 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 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 [E342] (ab32091)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-MEK1 antibody [E342] (AB32091)

Blocking/Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-MEK1 antibody [E342] (ab32091) at 1/5000 dilution

All lanes:

A431 (Human epidermoid carcinoma epithelial cell）whole cell lysate 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

Exposure time: 15s

• WB

Lab

Western blot - Anti-MEK1 antibody [E342] (AB32091)

Western blot : Anti-MAP2K1 antibody [E342] (ab32091) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32091 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 [E342] (ab32091) at 1/1000 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

false