All lanes : Anti-MEK3 + MEK6 antibody [EPR17340] (ab200831) at 1/1000 dilution

Lane 1 : Mouse spleen lysate at 10 µg
Lane 2 : Rat spleen lysate at 10 µg
Lane 3 : C6 (Rat glial tumor cells) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 6 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

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

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


Exposure time: 30 seconds

Blocking/Dilution Buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling MEK3 + MEK6 with ab200831 at 1/250 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and cytoplasmic staining on Mouse liver tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used 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.

Immunocytochemistry analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (human hepatocellular carcinoma) cells labeling MEK3 + MEK6 with ab200831 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear and cytoplasmic staining on HepG2 cell line is observed. The nuclear counterstain 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: ab200831 at 1/1000 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 + MEK6 antibody [EPR17340] (ab200831) at 1/10000 dilution + Human MEK6 recombinant protein fragment at 10 µg

Predicted band size: 39, 37 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Recombinant fragment of Human MEK6 protein contains aa139-334 with His-Tag®(22kDa).

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling MEK3 + MEK6 with ab200831 at 1/250 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and cytoplasmic staining on Human cervix carcinoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used 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.

All lanes : Anti-MEK3 + MEK6 antibody [EPR17340] (ab200831) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

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


Exposure time: 15 seconds

Blocking/Dilution Buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling MEK3 + MEK6 with ab200831 at 1/250 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and cytoplasmic staining on Human spleen tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used 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 + MEK6 antibody [EPR17340] (ab200831) at 1/10000 dilution + Human fetal liver lysate at 10 µg

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

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


Exposure time: 1 minute

Blocking/Dilution Buffer: 5% NFDM/TBST.

Flow cytometry analysis of HeLa cells labelling MEK3 + MEK6 (red) with purified ab200831 at dilution of 1/150. The secondary antibody used was Alexa Fluor^® 488 goat-anti-rabbit IgG (1/2000). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

MEK3 + MEK6 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab200831 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab200831 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 lysate 10ug (Input). Lane 2: ab200831 IP in HeLa whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200831 in Jurkat whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.