Anti-ACADM/MCAD antibody [EPR3708]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Flow cytometry overlay histogram showing wild-type U87-MG (green line) and ACADM/MCAD knockout U87-MG stained with ab92461 (magenta line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab92461) (1x 10⁶ in 100μl at 1 μg/ml (1/647)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control in U87-MG WT cells (black line) and U87-MG-ACADM/MCAD KO cells (grey line), at the same conditions as the primary antibody.

Acquisition of <5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ACADM/MCAD with purified ab92461 at 1 : 50 dilution (10 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunofluorescent staining of ACADM/MCAD in HeLa cells using ab92461 (unpurified) at 1/100 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

ab92461 (unpurified), at 1/100 dilution, staining ACADM/MCAD in formalin-fixed, paraffin-embedded Human liver tissue by immunohistochemistry.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunofluorescent analysis of 100% methanol-fixed 0.1% Triton X-100 permeabilized U-87 MG WT and U-87 MG ACADM KO cells labelling medium-chain specific acyl-CoA dehydrogenase with ab92461 at 1 μg/ml concentration, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). ab7291 Anti-alpha Tubulin antibody [DM1A] was used to counterstain tubulin at 1/1000 dilution (Magenta). The nuclear counterstain was DAPI (Blue).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ACADM/MCAD with unpurified ab92461 at 1/50 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ACADM/MCAD with purified ab92461 at 1/60 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

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Immunoprecipitation - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

ab92461 (purified) at 1/30 dilution (2 µg) immunoprecipitating ACADM/MCAD in Mouse heart lysate.
Lane 1 (input) : Mouse heart lysate 10 µg
Lane 2 (+) : ab92461 & Mouse heart lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab92461 in Mouse heart lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)

Predicted band size: 47 kDa

false

• WB

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Western blot - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

All lanes:

Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution

Lane 1:

Human heart lysate at 10 µg

Lane 2:

fetal liver lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

HepG2 cell lysate at 10 µg

Lane 5:

K562 cell lysate at 10 µg

Secondary

All lanes:

HRP conjugated Goat anti-Rabbit Ig at 1/2000 dilution

Predicted band size: 47 kDa

false

• WB

Lab

Western blot - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

All lanes:

Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Lane 2:

Human heart lysates at 20 µg

Lane 3:

Mouse heart lysates at 20 µg

Lane 4:

Rat heart lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 47 kDa

Observed band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ACADM/MCAD antibody [EPR3708] (AB92461)

All lanes:

Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 47 kDa

Observed band size: 43 kDa

false