Recombinant Anti-MEF2D antibody [EPR24993-10] (ab282731)

False colour image of Western blot: Anti-MEF2D antibody [EPR24993-10] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab282731 was shown to bind specifically to MEF2D. A band was observed at 67 kDa in wild-type HeLa cell lysates with no signal observed at this size in mef2d CRISPR-Cas9 edited cell line ab281636. The band observed in the CRISPR-Cas9 edited lysate lane below 67 kDa is likely to represent a truncated form of MEF2D. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and mef2d CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling MEF2D with ab282731 at 1/2000 (0.286 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse testis (PMID: 24694307). The section was incubated with ab282731 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling MEF2D with ab282731 at 1/50 (11.42 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green) Confocal image showing mainly nuclear staining in THP-1 cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) cells labelling MEF2D with ab282731 at 1/500 dilution (0.1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Low expression: SW480/ DLD-1 (PMID: 27364559).

Exposure time: 147 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

 Exposure time: 147 seconds

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling MEF2D with ab282731 at 1/2000 (0.286 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse cererbum (PMID: 28738418). The section was incubated with ab282731 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Blocking and diluting buffer and concentration: 5% NFDM/TBST

His-tagged human MEF2D recombinant protein (aa54-328) 10ng

Myc/DDDDK-tagged human MEF2A recombinant protein 10ng

Myc/DDDDK-tagged human MEF2B recombinant protein 10ng

Myc/DDDDK-tagged human MEF2C recombinant protein 10ng

 Exposure time: 10 seconds

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labelling MEF2D with ab282731 at 1/2000 (0.286 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat colon. The section was incubated with ab282731 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling MEF2D with ab282731 at 1/2000 (0.286 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat cererbum (PMID: 28738418). The section was incubated with ab282731 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling MEF2D with ab282731 at 1/50 (11.42 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green) Confocal image showing mainly nuclear staining in RAW 264.7 cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling MEF2D with ab282731 at 1/500 dilution (0.1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.