Anti-CPT1A antibody [EPR21843-71-2F]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Flow cytometry overlay histogram showing left wild-type HAP1 positive cells and right negative CPT1A knockout HAP1 stained with ab234111 (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 (ab234111) (1x 10⁶ in 100μl at 1 μg/ml (1/481)) 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 (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 525/40 bandpass filter.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized SK-OV-3 (human ovarian cancer cell line) cell line labeling CPT1A with ab234111 at 1/50 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue labeling CPT1A with ab234111 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) is observed. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CPT1A with ab234111 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; PMID : 15363638; PMID : 8679700) is observed. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Immunohistochemical analysis of paraffin-embedded human heart tissue labeling CPT1A with ab234111 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human heart is observed. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling CPT1A with ab234111 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular cytoplasmic staining in rat kidney (PMID : 18192268; PMID : 28956034; PMID : 15363638; PMID : 8679700) is observed. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling CPT1A with ab234111 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in mouse kidney (PMID : 18192268; PMID : 28956034; PMID : 15363638; PMID : 8679700) is observed. 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.

• WB

Lab

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Lanes 1- 2 : Merged signal (red and green). Green - ab234111 observed at 88 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab234111 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. ab234111 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 [EPR21843-71-2F] (ab234111) 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='/en-us/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 [EPR21843-71-2F] (AB234111)

Blocking and diluting buffer : 5% NFDM /TBST

Lanes 1 - 2:

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (ab234111) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/cpt1a-antibody-epr21843-71-2f-bsa-and-azide-free-ab235841'>ab235841</a>) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 1/20 dilution

Secondary

All lanes:

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

Observed band size: 88 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab234111 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. ab234111 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 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 [EPR21843-71-2F] (ab234111) at 1/1000 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='/en-us/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

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Blocking and diluting buffer : 5% NFDM/TBST

Lanes 1 - 3:

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/cpt1a-antibody-epr21843-71-2f-bsa-and-azide-free-ab235841'>ab235841</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (ab234111) 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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 88 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (AB234111)

Exposure time : Lanes 1 and 2 : 3 minutes; Lane 3 : 37 seconds; Lane 4 : 114 seconds; Lane 5 : 15 seconds; Lanes 6 and 7 : 92 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CPT1A antibody [EPR21843-71-2F] (ab234111) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor cell line) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 3:

Mouse kidney lysate at 20 µg

Lane 4:

Rat kidney lysate at 20 µg

Lane 5:

Human kidney lysate at 20 µg

Lane 6:

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

Lane 7:

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

Secondary

Lanes 1, 2, 3, 4, 6 and 7:

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 1/100000 dilution

Lane 5:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/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