In cells, excess of metabolic fuel is converted into fatty acids in cytosol and oxidized later in mitochondria to generate ATP and acetyl-CoA. In fatty acid synthesis, catalytic formation of malonyl-CoA (precursor for long-chain fatty acyl-CoA, LCFA-CoA) from acetyl-CoA by Acetyl-CoA carboxylase (ACC1) is the rate-limiting step. The translocation of LCFA-CoA from cytosol to mitochondria catalyzed by two carnitine palmitoyl transferases (CPT-1 & CPT-2) and regulated by ACC-2, is the rate-limiting step of mitochondrial fatty acid b-oxidation. Activities of ACC-1 and 2 are regulated by their phosphorylation by 5'-AMP-activated protein kinase (AMPK). Diabetes deranges AMPK master-switch and represses the ACC-1 gene-expression and stimulates excessive fatty acid oxidation, which in turn interferes with glucose metabolism. ACC-2 deficient mice accumulate 50% less fat in their adipose tissue suggesting that a pharmacological manipulation of ACC-2 may lead to loss of body fat in the context of normal caloric intake. Elevated levels of acyl-carnitines in arrhythmias and excessive oxidation of fatty acids in diabetes implicate both CPT-1 & CPT-2 as possible sites for pharmacological intervention.

ACC1 (rat 2345-aa, human 2345-aa, ~265 kDa, chromosome 17q21) is also known as ACC-alpha is a cytosolic enzyme, enriched in liver, adipose and lactating mammary tissues. ACC-1 from rat, human, chicken are over 90% identical. ACC1 catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, the rate-limiting step in the biogenesis of LCFA-CoA. ACC1 carries three functions: biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase (catalytic activity). Two variants of ACC-1 have been described: one with 8 additional amino acids commencing at Pro-1196, and the other which is 59 aa shorter than the predominant fat and liver isoform exist in mammals. The presence of 8 additional amino acids inhibits the in vitro phosphorylation of the Ser1200 by camp-dependent kinase. The two ACC1 isoform are differentially regulated in a tissue specific manner and under different physiological conditions. The activity of ACC1 is finely regulated by hormone dependent phosphorylation and dephosphorylation. ACC-2 (rat 2456-aa, human 2483-aa, ~280 kDa, chromosome 12q24.1), also known as ACC-beta, is predominantly present in heart and skeletal muscle and to a lesser extent in liver. An additional

ACC-2 isoform (270 kDa) is present in liver. In contrast to ACC-1, which is cytosolic and catalyzes only fatty acid synthesis, ACC-2 co-localizes with CPT-1 in the 'contact sites' of the mitochondrial membranes and regulates mitochondrial fatty acid oxidation as well by inhibiting CPT-1 by its product malonyl-CoA. ACC-2 contains an unique 114-aa long N-terminus peptide, accounting in part, for its regulatory role in fatty acid oxidation. ACC2 deficient mice accumulate 10-30 fold less malonyl-CoA in heart and muscle and show 50% less fat in the adipose tissue.

Mitochondrial oxidation of LC-FCA is initiated by the sequential action of CPT-1, which is located in the outer membrane, and CPT-2, which is located in the inner membrane together with a carnitine-acylcarnitine translocase. CPT-1 liver or CPT1A or LCPT-1 (mouse 764-aa, rat 773-aa, human 773-aa, ~88-kda, chromosome 11q13) is malonyl-CoA-sensitive enzyme localized on the outer surface of mitochondrial 'contact sites'. It catalyzes the conversion of long-chain acyl-CoA into acyl-carnitine, committing the acyl moiety to intramitochondrial oxidation. It is predominantly expressed in kidney, liver and in trace amounts in heart. The 'muscle' isoform CPT1B or CPT1M or MCPT-1 (mouse/rat/human 772-aa, chromosome 22q13.3) is found in heart, skeletal muscle, adipose tissue and brain. The aa sequences of the two isoforms are ~61% identical.

CPT-2 (mouse/rat/human 658-aa, ~74 kDa, chromosome 1p32, ~20% identity with CPT1) is a ubiquitous malonyl-CoA-insensitive transferase localized in the inner mitochondrial membrane. It catalyzes the re-synthesis of acyl-CoA from acyl-carnitines. CPT-2 deficiency leads to the most commonly inherited, lipid myopathy in adults characterized by exercise-induced pain, stiffness, and myoglobinuria.

AMPK is a heterotrimer of alpha, beta, and gamma subunits. Coexpression of all three subunit is required for kinase activity. The enzyme plays a key role in carbohydrate and fat metabolism by phosphorylating various target proteins including ACC-1 and ACC-2. AMPK-alpha 1 expression is poor in kidney, liver, lung, heart and brain; whereas AMPK-alpha 2 is predominantly expressed in skeletal and heart muscles and liver. Multiple isoform of AMPK-Beta 1-2 and gamma 1-3 have been reported.
 

M= Mouse; R=Rat; H=Human; Ha=Hamster; Rb=Rabbit; B=Bovine; CT= near C-terminus; NT=near N-terminus; Internal=Middle of protein.

m=mouse; r=rat; h=human; b=bovine; d=dog; ~CT or ~NT=near C or N-terminus. EC=Extracellular; CP=Cytoplasmic domain; Control peptides (unconjugated, free, antigenic peptides), because of their small size, are not recommended for Western. They should be used in ELISA/antibody blocking studies.

* Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visualization with antibodies.