Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: MEK3 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab195037 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab195037 was shown to specifically react with MEK3 when MEK3 knockout samples were used. Wild-type and MEK3 knockout samples were subjected to SDS-PAGE. ab195037 and ab8245 (loading control to GAPDH) were both diluted to 1/5000 and 1/10000 respectively 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nucleus staining on Human skeletal muscle is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

 

1:Ben-Levy R,et.al.(1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol, 8(19):1049-1057.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling MEK3 with ab195037 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing both nuclear and cytoplasmic staining on NIH/3T3 cell line.  The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab195037 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/50000 dilution + HepG2 (Human liver hepatocellular carcinoma) whole cell lysates at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 39 kDa
Observed band size: 34,39 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

Flow Cytometry analysis of NIH/3T3 (mouse embryo) labelling MEK3 with purified ab195037 at 1/1000 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Alexa Fluor® 488 goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

 

All lanes : Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 39 kDa
Observed band size: 34,39 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

Immunohistochemical analysis of paraffin-embedded Human lung adenocarcinoma tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on cancer cells of Human lung adenocarcinoma is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/5000 dilution + Human skeletal muscle lysates at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 39 kDa
Observed band size: 34,39 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

All lanes : Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/10000 dilution

Lane 1 : Mouse spleen lysates
Lane 2 : Rat spleen lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 39 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on mouse skeletal muscle is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on rat skeletal muscle is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

MEK3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab195037 at 1/90 dilution. Western blot was performed from the immunoprecipitate using ab195037 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.

 

Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.