Anti-CPT1A 抗体 [EPR21843-71-1C]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and CPT1A knockout HAP1 stained with ab220789 (magenta line). The cells were fixed with 4% formaldehyde (10 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 (ab220789) (1x 10⁶ in 100μl at 0.2 μg/ml (1/2970)) for 30min at 22°C.

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

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control in HAP1 WT cells (black line) and HAP1-CPT1A KO cells (grey line), at the same conditions as the primary antibody.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in wild-type A-549 fixed with 80% methanol (5 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-CPT1A antibody [EPR21843-71-1C] (AB220789)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling CPT1A with ab220789 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue labeling CPT1A with ab220789 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular cytoplasmic staining in human ovarian carcinoma (PMID : 26716645). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-OV-3 (human ovarian cancer cell line) cells labeling CPT1A with ab220789 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mitochondrial staining in SK-OV-3 cell line.

The nuclear counter stain is DAPI (blue). COX IV is detected with Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (red).

The negative controls are as follows :

-ve control 1 : ab220789 at 1/100 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling CPT1A with ab220789 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mitochondrial staining in HeLa cell line.

The nuclear counter stain is DAPI (blue). COX IV is detected with Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (red).

The negative controls are as follows :

-ve control 1 : ab220789 at 1/100 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CPT1A with ab220789 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular cytoplasmic staining in human kidney (PMID : 18192268; PMID : 28956034). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Immunofluorescent analysis of 100% methanol-fixed-fixed 0.1%, Triton X-100 permeabilized Hap1 WT and Hap1-CPT1A KO cells labelling CPT1A protein with ab220789 at 0.2 μg/ml concentration followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 μg/ml dilution (Green). Image showing mitochodrial staining in Hap1 WT cell line. ab7291 Anti-alpha Tubulin antibody [DM1A] was used to counterstain tubulin at 1/1000 dilution (Magenta). The nuclear counterstain was DAPI (Blue).

• IP

Supplier Data

Immunoprecipitation - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

CPT1A was immunoprecipitated from 0.35 mg of SK-OV-3 (human ovarian cancer cell line) whole cell lysate with ab220789 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220789 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : SK-OV-3 whole cell lysate 10 μg (Input).

Lane 2 : ab220789 IP in SK-OV-3 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220789 in SK-OV-3 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789)

Predicted band size: 88 kDa

Observed band size: 88 kDa

false

• WB

Lab

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Lanes 1- 2 : Merged signal (red and green). Green - ab220789 observed at 88 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab220789 was shown to react with CPT1A in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266319 (knockout cell lysate ab256880) was used. Wild-type HEK-293T and CPT1A knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab220789 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CPT1A knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CPT1A knockout HEK-293T cell line (<a href='/products/cell-lines/human-cpt1a-knockout-hek-293t-cell-line-ab266319'>ab266319</a>)

Predicted band size: 88 kDa

Observed band size: 88 kDa

false

• WB

Supplier Data

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab220789 was shown to specifically react with CPT1A in wild-type HAP1 cells as signal was lost in CPT1A knockout cells. Wild-type and CPT1A knockout samples were subjected to SDS-PAGE. ab220789 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/5000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CPT1A knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

SK-OV-3 (human ovarian cancer cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 88 kDa

Observed band size: 88 kDa

true

Exposure time: 92s

• WB

Lab

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Blocking and diluting buffer and concentration : 5% NFDM /TBST. CPT1A is strongly expressed in kidney and heart, and lower in liver and skeletal muscle. We recommend loading higher amount of lysate or using lower antibody dilution to detect signal in HepG2 lysate.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate at 20 µg

Lane 3:

HepG2 (Human hepatocellular carcinoma epithelial cell) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 88 kDa

Observed band size: 88 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. CPT1A is strongly expressed in kidney and heart, and lower in liver and skeletal muscle. We recommend loading higher amount of lysate or using lower antibody dilution to detect signal in liver lysate.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 88 kDa

Observed band size: 88 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (AB220789)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1:

His-tagged human CPT1A recombinant protein (aa406-755) at 0.02 µg

Lane 2:

His-tagged human CPT1B recombinant protein (aa407-756) at 0.02 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 88 kDa

Observed band size: 38 kDa

true

Exposure time: 10s