Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ATP citrate lyase knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab40793 observed at 125 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab40793 was shown to specifically react with ATP citrate lyase in wild-type HAP1 cells as signal was lost in ATP citrate lyase knockout cells. Wild-type and ATP citrate lyase knockout samples were subjected to SDS-PAGE. ab40793 and ab9484 (Mouse anti-GAPDH 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 HeLa cells labelling ATP citrate lyase with purified ab40793 at 1/50. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human clear cell carcinoma of kidney tissue sections labeling ATP citrate lyase with Purified ab40793 at 1:100 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ATP citrate lyase with purified ab40793 at 1:30 dilution (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 - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-ATP citrate lyase antibody [EP704Y] (ab40793) at 1/50000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) 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: 122 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

ab40793 (purified) at 1:20 dilution (1.5μg) immunoprecipitating ATP citrate lyase in HeLa whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate,10μg
Lane 2 (+): ab40793 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40793 in HeLa whole cell lysate. 

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-ATP citrate lyase antibody [EP704Y] (ab40793) at 1/10000 dilution (purified)

Lane 1 : Rat lung lysates
Lane 2 : C6 (Rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 122 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-ATP citrate lyase antibody [EP704Y] (ab40793) at 1/5000 dilution (unpurified) + HeLa cell lysate

Predicted band size: 122 kDa
Observed band size: 122 kDa

Overlay histogram showing HeLa cells stained with unpurified ab40793 (red line). The cells were fixed with 80% methanol (5 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 (ab40793, 1/100 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) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Unpurified ab40793 at 1/40 immunoprecipitating ATP citrate lyase in HeLa (human cervix adenocarcinoma) whole cell lysate.

Lane 1 (input): HeLa (human cervix adenocarcinoma) whole cell lysate 10μg

Lane 2 (+): ab40793 + HeLa (human cervix adenocarcinoma) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40793 in HeLa (human cervix adenocarcinoma) whole cell lysate

For western blotting, ab40793 at 1/1000 dilution and ab131366 VeriBlot for IP (HRP) was used for detection at 1/10000.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.