All lanes : Anti-MEK1 + MEK2 antibody [EPR16667] (ab178876) at 1/20000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : MAP2K2 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 43, 44 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).

ab178876 staining MEK1 + MEK2 in the human cell line HeLa (human cervix adenocarcinoma) by 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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).

Clone EPR16667 (ab215263) has been successfully conjugated by Abcam. This image was generated using Anti-MEK1 + MEK2 antibody [EPR16667] (Alexa Fluor® 488). Please refer to ab200177 for protocol details.

ab200177 staining MEK1 + MEK2 in A431 cells. The cells were fixed with 4% formaldehyde (10 min), 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 overnight at +4°C with ab200177 at a 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EPR16667 (ab215263) has been successfully conjugated by Abcam. This image was generated using Anti-MEK1 + MEK2 antibody [EPR16667] (PE). Please refer to ab225265 for protocol details.

Overlay histogram showing A431 cells stained with ab225265 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225265, 1/1000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

This antibody gave a positive signal in A431 cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).

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

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).

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

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).

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

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178876).