All lanes : Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CPT1A knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 88 kDa

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.

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).

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.

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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CPT1A knockout HAP1 whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : SK-OV-3 (human ovarian cancer cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa
Observed band size: 88 kDa


Exposure time: 92 seconds

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.

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.

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.

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).

All lanes : Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : Human kidney lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa
Observed band size: 88 kDa

Exposure time : Lane 1: 3 minutes; Lane 2: 8 seconds; Lane 3: 10 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-CPT1A antibody [EPR21843-71-1C] (ab220789) at 1/1000 dilution

Lane 1 : His-tagged human CPT1A recombinant protein (aa406-755)
Lane 2 : His-tagged human CPT1B recombinant protein (aa407-756)

Lysates/proteins at 0.02 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.