Anti-ATP citrate lyase antibody [EP704Y]

10 product images

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab40793 in HeLa whole cell lysate.
  
  For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1:1000 dilution.
  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  Predicted band size: 120 kDa

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  Western blot - 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, Anti-GAPDH antibody [mAbcam 9484] - 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 Anti-GAPDH antibody [mAbcam 9484] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  Predicted band size: 120 kDa

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab40793 in HeLa (human cervix adenocarcinoma) whole cell lysate

  For western blotting, ab40793 at 1/1000 dilution and VeriBlot for IP Detection Reagent (HRP) ab131366 VeriBlot for IP (HRP) was used as the secondary antibody at 1/10000.

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  Predicted band size: 120 kDa

  Observed band size: 122 kDa

  Exposure time: 10s

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  Flow Cytometry (Intracellular) - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  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).

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

  Blocking and diluting buffer: 5% NFDM/TBST.

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

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

  Secondary

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

  Predicted band size: 120 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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

  Blocking and diluting buffer: 5% NFDM/TBST.

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

  Lane 1: Rat lung lysates at 15 µg

  Lane 2: C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

  Secondary

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

  Predicted band size: 120 kDa

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

  All lanes: Western blot - Anti-ATP citrate lyase antibody [EP704Y] (ab40793) at 1/5000 dilution

  All lanes: HeLa cell lysate

  Predicted band size: 120 kDa

  Observed band size: 122 kDa

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  Flow Cytometry (Intracellular) - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  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) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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.
  

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  Immunocytochemistry/ Immunofluorescence - Anti-ATP citrate lyase antibody [EP704Y] (ab40793)

  Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ATP citrate lyase with purified ab40793 at 1/50. Cells were fixed with 100% methanol. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.