Figure 1: CPT1A protein structure.

CPT1A target introduction

Protein Function

• Carnitine palmitoyltransferase 1A (CPT1A) catalyzes the transfer of acyl groups from long-chain fatty acyl-CoA compounds to carnitine, which is an essential step in the uptake of long-chain fatty acids and subsequent beta-oxidation in mitochondria.

Protein Expression

• High expression in the kidney and heart, lower expression in the liver and skeletal muscle.

Protein Localization

• Located in the outer mitochondrial membrane.

Figure 2: CPT1A experimental result image, Immunocytochemistry/ Immunofluorescence - Anti-CPT1A antibody [EPR21843-71-1C] (ab220789). Green: CPT1A; Red: COX IV (mitochondrial marker); Blue: DAPI.

Isoforms & Post-translational modifications

• Human (P50416): Isoforms 1-2, 86-88 kDa (predicted)
• Mouse (P97742): 88 kDa (predicted)
• Rat (P32198): 88 kDa (predicted)
• Existence of phosphorylation, acetylation, nitration, and other modification sites.

WB experiment tips

Precautions

• CPT1A is a multi-pass transmembrane protein. If the band molecular weight becomes larger, fuzzy, or trailing after boiling the sample, we recommend not boiling the sample.
• CPT1A is located on the outer membrane of mitochondria. If the signal weakens the whole cell lysate, we recommend extracting mitochondrial components to enhance the detection signal.
• CPT1A has expression characteristics and is expressed at a low level in liver tissue. Suppose samples with weak target protein content (e.g. HepG2, liver tissue) are detected. In that case, we recommended increasing the loading amount, reducing the antibody dilution ratio to improve the detection signal, and increasing the positive control.

Positive control

• HAP1 whole cell lysate, HeLa whole cell lysate, SK-OV-3 whole cell lysate
• Mouse, rat, human kidney tissue lysate

Example of results

Figure 3: Anti-CPT1A antibody [EPR21843-71-2F] (ab234111).

Lane 1: 20 µg C6 whole cell lysate
Lane 2: 20 µg MCF7 whole cell lysate
Lane 3: 20 µg mouse kidney tissue lysate
Lane 4: 20 µg rat kidney tissue lysate
Lane 5: 20 µg human kidney tissue lysate
Lane 6: 20 µg HeLa whole cell lysate
Lane 7: 20 µg SK-OV-3 whole cell lysate

Predicted band size: 88 kDa
Detected band size: 88 kDa

Figure 4: Anti-CPT1A antibody [EPR21843-71-2F] (ab234111).

Lane 1: 20 µg of human kidney tissue lysate.
Lane 2: 20 µg of human liver tissue lysate.

Predicted band size: 88 kDa.
Detected band size: 88 kDa.

Figure 5: Anti-CPT1A antibody [EPR21843-71-2F] (ab234111).

Lane 1: 20 µg MCF7 whole cell lysate.
Lane 2: 20 µg HeLa whole cell lysate.
Lane 3: 20 µg HepG2 whole cell lysate.

Predicted band size: 88 kDa.
Detected band size: 88 kDa.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex proteinase inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Transferring:

1. We recommend using Ponceau S staining after transfer to check for successful transfer.

References

1. L IJlst, H Mandel, W Oostheim, J P Ruiter, A Gutman, R J Wanders. Molecular basis of hepatic carnitine palmitoyltransferase I deficiency. Case Reports J Clin Invest. 1998 Aug 1;102(3):527-31. doi: 10.1172/JCI2927.
2. C Prip-Buus, L Thuillier, etc. Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community. Mol Genet Metab. 2001 May;73(1):46-54. doi: 10.1006/mgme.2001.3176.
3. Stéphanie Gobin, Laure Thuillier, etc. Functional and structural basis of carnitine palmitoyltransferase 1A deficiency. J Biol Chem. 2003 Dec 12;278(50):50428-34. doi: 10.1074/jbc.M310130200. Epub 2003 Sep 29.