All lanes : Anti-MEF2A + MEF2C antibody [EPR19089-34] (ab197070) at 1/1000 dilution

Lane 1 : Recombinant Human MEF2A protein (ab152519)
Lane 2 : Human MEF2C full length protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 51 kDa
Observed band size: 55,83 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

ab197070 recognizes the full length tagged recombinant proteins MEF2A and MEF2C which have expected molecular weights of 83 and 55 kDa respectively.

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 2% Bovine Serum Albumin before being incubated with ab197070 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Flow Cytometry analysis of Raji(human Burkitt's lymphoma) labelling MEF2A + MEF2C with purified ab197070 at 1/30 (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-MEF2A + MEF2C antibody [EPR19089-34] (ab197070) at 1/1000 dilution

Lane 1 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

MEF2C is expressed specifically in cells from B-lymphocyte lineage but not T cell lineage. Jurkat cell lysate serves as a negative control (PMID: 9798649).

The 50-60KD bands observed are consistent with what has been described in the literature (PMID: 18450586)

All lanes : Anti-MEF2A + MEF2C antibody [EPR19089-34] (ab197070) at 1/5000 dilution

Lane 1 : Mouse cerebral cortex lysate
Lane 2 : Rat cerebral cortex lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The 50-60KD bands observed are consistent with what has been described in the literature (PMID: 18450586)

All lanes : Anti-MEF2A + MEF2C antibody [EPR19089-34] (ab197070) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 1minute; Lane 2 and 3: 3minutes.

The 50-60KD bands observed are consistent with what has been described in the literature (PMID: 18450586).

All lanes : Anti-MEF2A + MEF2C antibody [EPR19089-34] (ab197070) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : Raw264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 15 seconds; Lane 2: 30 seconds.

The 50-60KD bands observed are consistent with what has been described in the literature (PMID: 18450586).

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on B lymphocytes of the Human tonsil 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.

Immunohistochemical analysis of paraffin-embedded Human colonic adenocarcinoma tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on lymphocytes of the colonic adenocarcinoma 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.

Immunohistochemical analysis of paraffin-embedded Human thymus tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on normal Human thymus is observed. Counter stained with Hematoxylin.

 

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

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on B lymphocytes of the mouse spleen, the T cells in the periarterial lymphatic sheath showed negative staining. 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.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on the mouse colon epithelium, the lymphocytes on the interstitial substance showed nuclear staining. 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.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on B lymphocytes of the rat spleen, the T cells in the periarterial lymphatic sheath showed negative staining. 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.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling MEF2C with ab197070 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on rat testis is observed. Counter stained with Hematoxylin.

 

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 K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling MEF2C with ab197070 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on K562 cell line. 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 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab197070 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 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 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (Human Burkitt's lymphoma cell line) cells labeling MEF2C with ab197070 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Raji cell line. 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 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab197070 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 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 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (Human Burkitt's lymphoma cell line) and Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling MEF2C with ab197070 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Raji cell line. Negative expression in Jurkat cells. 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 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab197070 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 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 1/1000 dilution.