All lanes : Anti-Acetyl Coenzyme A Carboxylase antibody [EPR4971] (ab109368) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ACACA (Acetyl Coenzyme A Carboxylase) knockout HAP1 whole cell lysate
Lane 3 : Hela whole cell lysate
Lane 4 : A431 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 266 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab109368 observed at 265 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab109368 was shown to specifically react with Acetyl Coenzyme A carboxylase in wild-type HAP1 cells as signal was lost in ACACA (Acetyl Coenzyme A Carboxylase) knockout cells. Wild-type and ACACA (Acetyl Coenzyme A Carboxylase) knockout samples were subjected to SDS-PAGE. Ab109368 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of 293 (Human embryonic kidney epithelial cell) cells labeling Acetyl Coenzyme A carboxylase with Purified ab109368 at 1:250 dilution (2.1μg/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). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Acetyl Coenzyme A carboxylase with purified ab109368 at 1:100 dilution (5 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - 90% methanol. Unlabeled control - Rabbit monoclonal IgG (Black). Cells without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling Acetyl Coenzyme A carboxylase with purified  ab109368 at 1:500 dilution (1.05 μg/ml). Heat mediated antigen retrieval was performed using citrate Buffer, pH6.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Overlay histogram showing SH-SY5Y cells stained with unpurified ab109368 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109368, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Acetyl Coenzyme A Carboxylase antibody [EPR4971] (ab109368) at 1/1000 dilution (unpurified)

Lane 1 : 293T cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : SH-SY5Y cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 266 kDa

Immunofluorescent staining of 293 cells using unpurified ab109368 at 1/100 dilution

All lanes : Anti-Acetyl Coenzyme A Carboxylase antibody [EPR4971] (ab109368) at 1/5000 dilution

Lane 1 : 293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 266 kDa
Observed band size: 266 kDa

5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded skeletal muscle tissue using unpurified ab109368 at 1/250 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded brain tissue using unpurified ab109368 at 1/250 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.