Nori Mouse CPT-1 ELISA Kit Summary

Alternative names for CPT-1: Carnitine palmitoyl transferase I (CPT1), carnitine acyltransferase I, CPTI, CAT1, CoA:carnitine acyl transferase (CCAT), palmitoyl CoA transferase I, CPT1A

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Carnitine palmitoyl transferase I (CPT1) also known as carnitine acyltransferase I, CPTI, CAT1, CoA:carnitine acyl transferase (CCAT), or palmitoyl CoA transferase I, is a mitochondrial enzyme responsible for the formation of acyl carnitines by catalyzing the transfer of the acyl group of a long-chain fatty acyl-CoA from coenzyme A to l-carnitine. It is part of a family of enzymes called carnitine acyltransferases.^[1] Palmitoyl carnitine is the substrate for CPT1, but other fatty acids may also be substrates.^[2] CPT1 allows movement of the acyl carnitine from the cytosol into the intermembrane space of mitochondria. CPT1 has three isoforms such as CPT1A, CPT1B, and CPT1C. CPT1 is associated with the outer mitochondrial membrane and with type 2 diabetes and insulin resistance. Its role in fatty acid metabolism makes CPT1 important in many metabolic disorders such as diabetes. CPT I is the first component and rate-limiting step of the carnitine palmitoyl transferase system, catalyzing the transfer of the acyl group from coenzyme A to carnitine to form palmitoyl carnitine. A translocase then shuttles the acyl carnitine across the inner mitochondrial membrane where it is converted back into palmitoyl-CoA. By acting as an acyl group acceptor, carnitine may also play the role of regulating the intracellular CoA:acyl-CoA ratio.^[3] CPT1 is inhibited by malonyl-CoA, although the exact mechanism of inhibition remains unknown. The CPT1 skeletal muscle and heart isoform, CPT1B, has been shown to be 30-100-fold more sensitive to malonyl-CoA inhibition than CPT1A. This inhibition is a good target for future attempts to regulate CPT1 for the treatment of metabolic disorders.^[4] The “CPT1A” form is associated with carnitine palmitoyl transferase I deficiency.^[5] This rare disorder confers risk for hepatic encephalopathy, hypoketotic hypoglycemia, seizures, and sudden unexpected death in infancy.^[6] The increased levels of malonyl-CoA caused by hyperglycemia and hyperinsulinemia inhibit CPT1, which causes a subsequent decrease in the transport of long chain fatty acids into muscle and heart mitochondria, decreasing fatty acid oxidation in such cells. The shunting of LCFAs away from mitochondria leads to the observed increase in FFA levels and the accumulation of fat in skeletal muscle.^[7]

References

1. Jogl G, Tong L (2003). Cell. 112 (1): 113–22.
2. Bonnefont JP, et al. (2004). Molecular Aspects of Medicine. 25 (5–6): 495–520.
3. Jogl G, et al. (2004). Annals of the New York Academy of Sciences. 1033 (1): 17–29.
4. Shi J, et al. (2000). Biochemistry. 39 (4): 712–717.
5. Ogawa E, et al. (2002). Journal of Human Genetics. 47 (7): 342–7.
6. Collins SA, et al. (2010). Molecular Genetics and Metabolism. 101 (2–3): 200–204.
7. Rasmussen BB, et al. (2002). The Journal of Clinical Investigation. 110 (11): 1687–93.