All lanes : Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (Purified)

Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
Lane 2 : Human heart lysates
Lane 3 : Mouse heart lysates
Lane 4 : Rat heart lysates

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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.

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.

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).

Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (Purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 47 kDa
Observed band size: 43 kDa why is the actual band size different from the predicted?

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.

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.

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.

All lanes : Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (unpurified)

Lane 1 : Human heart lysate
Lane 2 : fetal liver lysate
Lane 3 : HeLa cell lysate
Lane 4 : HepG2 cell lysate
Lane 5 : K562 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated Goat anti-Rabbit Ig at 1/2000 dilution

Predicted band size: 47 kDa

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.

 

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

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.