Malonyl-CoA is a coenzyme A derivative. It plays a key role in chain elongation in fatty acid biosynthesis and polyketide biosynthesis. Malonyl-CoA is also used in transporting alpha-ketoglutarate across the mitochondrial membrane into the mitochondrial matrix. Malonyl-CoA is formed by carboxylating acetyl-CoA using the enzyme acetyl-CoA carboxylase. One molecule of acetyl-CoA joins with a molecule of carbon dioxide, requiring energy rendered from ATP. Malonyl-CoA is utilised in fatty acid biosynthesis by the enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT). MCAT serves to transfer malonate from malonyl-CoA to the terminal thiol of holo-acyl carrier protein (ACP). Controversy still exists as to whether MCAT is also involved in bacterial polyketide biosynthesis, however there is evidence that the acyl carrier protein from a variety of bacterial polyketide synthases is capable of self-malonylation in the presence of malonyl-CoA. Malonyl-CoA is a highly-regulated molecule in fatty acid synthesis; as such, it inhibits the rate-limiting step in beta-oxidation of fatty acids. Malonyl CoA inhibits coenzyme A from associating with carnitine, thereby preventing coenzyme A from entering the mitochondria where fatty acid oxidation and degradation occur.
<PRINCIPLE OF THE ASSAY>
The microtiter plate provided in this kit has been pre-coated with an antibody specific to malonyl CoA. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for malonyl CoA and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain malonyl CoA, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of malonyl CoA in the samples is then determined by comparing the O.D. of the samples to the standard curve.
This assay recognizes recombinant and natural bovine Malonyl CoA. No significant cross-reactivity or interference was observed.
0.625 ng/ml-40 ng/ml.
The standard curve concentrations used for the ELISA’s were 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625 ng/ml.
The minimum detectable dose of bovine Malonyl CoA is typically less than 0.156 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero
|3.||Sample Diluent||1 x 20 ml|
|4.||Biotin-antibody Diluent||1 x 10 ml|
|5.||HRP-avidin Diluent||1 x 10 ml|
|6.||Biotin-antibodyt||1 x 120μl|
|7.||HRP-avidin||1 x 120μl|
|8.||Wash Buffer||1 x 20 ml (25×concentrate)|
|9.||TMB Substrate||1 x 10 ml|
|10.||Stop Solution||1 x 10 ml|