Anti-ATP citrate lyase antibody [EP704Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ATP citrate lyase antibody [EP704Y] (AB40793)

Flow cytometry overlay histogram showing left wild-type HAP1 positive cells and right negative ACLY knockout HAP1 stained with ab40793 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab40793) (1x 10⁶ in 100μl at 0.04 μg/ml (1/6225 dilution)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in wild-type HAP1 fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

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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).

• IHC-P

Lab

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 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.

• ICC/IF

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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. 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.

• Flow Cyt (Intra)

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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) (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.

• IP

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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 (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:

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

Predicted band size: 120 kDa

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• IP

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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 (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 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

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Exposure time: 10s

• WB

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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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• WB

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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, 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.

All lanes:

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

Predicted band size: 120 kDa

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• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 120 kDa

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• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 120 kDa

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