Anti-MEK2 antibody [Y78]

• WB

Lab

Western blot - Anti-MEK2 antibody [Y78] (AB32517)

Western blot : Anti-MAP2K2 antibody [Y78] (ab32517) 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, ab32517 was shown to bind specifically to MAP2K2. A band was observed at 44 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MAP2K2 knockout cell line. To generate this image, wild-type and MAP2K2 knockout HCT 116 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-MEK2 antibody [Y78] (ab32517) at 1/10000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human MAP2K2 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-map2k2-knockout-hct116-cell-line-ab286599'>ab286599</a>)

Lane 2:

MAP2K2 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T ab255553 cell lysate at 20 µg

Lane 4:

MAP2K2 knockout HEK-293T ab261003 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 44 kDa

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MEK2 antibody [Y78] (AB32517)

Overlay histogram showing HeLa cells stained with ab32517 (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 (ab32517, 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MEK2 antibody [Y78] (AB32517)

ab32517 staining MEK2 in wild-type HAP1 cells (top panel) and MEK2 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32517 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK2 antibody [Y78] (AB32517)

Immunohistochemical analysis of paraffin embedded human prostate carcinoma using ab32517 at a dilution of 1/250.

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MEK2 antibody [Y78] (AB32517)

Immunofluorescent staining of HeLa cells using ab32517 at a dilution of 1/250.

• WB

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Western blot - Anti-MEK2 antibody [Y78] (AB32517)

All lanes:

Western blot - Anti-MEK2 antibody [Y78] (ab32517) at 1/10000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 44 kDa

Observed band size: 45 kDa

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• WB

Lab

Western blot - Anti-MEK2 antibody [Y78] (AB32517)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab32517 overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) at a 1 : 10000 dilution for 1hr at room temperature and then imaged.

All lanes:

Western blot - Anti-MEK2 antibody [Y78] (ab32517) at 1/10000 dilution

Lane 1:

Jurkat Whole Cell Lysate at 20 µg

Lane 2:

Mouse Brain Tissue Lysate at 20 µg

Lane 3:

Mouse Lung Tissue Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa

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• WB

Lab

Western blot - Anti-MEK2 antibody [Y78] (AB32517)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : MEK2 knockout HAP1 cell lysate (20 μg)
Lane 3 : Jurkat cell lysate (20 μg)
Lane 4 : K562 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab32517 observed at 44 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32517 was shown to specifically react with MEK2 when MEK2 knockout samples were used. Wild-type and MEK2 knockout samples were subjected to SDS-PAGE. ab32517 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-MEK2 antibody [Y78] (ab32517)

Predicted band size: 44 kDa

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• WB

Lab

Western blot - Anti-MEK2 antibody [Y78] (AB32517)

Lanes 1 - 2 : Merged signal (red and green). Green - ab32517 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32517 was shown to react with MEK2 in wild-type HEK-293T cells in western blot with loss of signal observed in MAP2K2 knockout cell line ab266315 (MAP2K2 knockout cell lysate ab257512). 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 ab32517 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 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-MEK2 antibody [Y78] (ab32517) at 1/10000 dilution

Lane 1:

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

Lane 2:

MAP2K2 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human MAP2K2 (MEK2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-map2k2-mek2-knockout-hek-293t-cell-line-ab266315'>ab266315</a>)

Predicted band size: 44 kDa

Observed band size: 45 kDa

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