Recombinant Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

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.

Intracellular 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).

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.

Blocking and diluting buffer: 5% NFDM/TBST.

Overlay histogram showing HeLa cells stained withunpurified 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.