All lanes : Anti-MEK3 + MEK6 antibody [EPR17345] (ab181555) at 1/50000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysates
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 37, 39 kDa
Observed band size: 39,37,34 kDa why is the actual band size different from the predicted?

Based on the sequence analysis, ab181555 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms and MEK6.

 

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling MEK3 + MEK6 with ab181555 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm and nuclear staining on HeLa cell line is observed. The nuclear counterstain 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. ab181555 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 1/400 dilution.

Anti-MEK3 + MEK6 antibody [EPR17345] (ab181555) at 1/10000 dilution + Human MEK6 recombinant protein fragment at 10 µg

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

Predicted band size: 37, 39 kDa
Observed band size: 22 kDa why is the actual band size different from the predicted?

Recombinant protein covers 139aa-334aa with a His tag (22kDa).

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-MEK3 + MEK6 antibody [EPR17345] (ab181555) at 1/5000 dilution

Lane 1 : Rat liver lysates
Lane 2 : Mouse liver lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 37, 39 kDa
Observed band size: 39,37,34 kDa why is the actual band size different from the predicted?

Based on the sequence analysis, ab181555 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms and MEK6.

 

Blocking/Dilution buffer: 5% NFDM/TBST.

 

All lanes : Anti-MEK3 + MEK6 antibody [EPR17345] (ab181555) at 1/2000 dilution

Lane 1 : Mouse heart lysates
Lane 2 : Mouse kidney lysates
Lane 3 : Rat heart lysates
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 5 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 37, 39 kDa
Observed band size: 39,37,34 kDa why is the actual band size different from the predicted?

Based on the sequence analysis, ab181555 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms and MEK6.

 

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling MEK3 + MEK6 with ab181555 at 1/100 dilution followed by ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. Cytoplasmic staining on Human skeletal muscle is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary ab.

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

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling MEK3 + MEK6 with ab181555 at 1/100 dilution followed by ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. Cytoplasmic staining on Mouse skeletal muscle is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary ab.

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 + MEK6 with ab181555 at 1/100 dilution followed by ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. Cytoplasmic staining on Rat skeletal muscle is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary ab.

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

MEK3 + MEK6 were immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181555 at 1/140 dilution. Western blot was performed from the immunoprecipitate using ab181555 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.

MEK3 + MEK6 were immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181555 at 1/140 dilution. Western blot was performed from the immunoprecipitate using ab33866 (Rabbit monoclonal [EP557Y] to MEK6) 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.