The Immunogen: Adenylate cyclases are enzymes which interact with and are activated by the GTP bound alpha subunits of trimeric G-proteins. Activated adenylate cyclases are responsible for the production of the important "second messenger" signalling molecule cyclic-AMP, which is generated from ATP. The type III adenylate cyclase enzyme is localized in the membranes surrounding the cilia in neurons, and our antibody is an excellent marker of neuronal cilia in the brain and in cells in tissue culture. Adenylate cyclase type III is a large complex molecule of, in the human, 1145 amino acids with a deduced molecular weight of 129kDa. The protein may be variably glycosylated, so that on SDS-PAGE and western blots it runs as a diffuse band of about 160kDa in cortex and about 200kDa in olfactory epithelium (see below). The molecule has a complex structure, with 12 transmembrane domains and two cyclase domains. Each cyclase domain is immediately C-terminal to 6 transmembrane segments, but only the second, C-terminal cyclase is believed to be catalytically active. Our antibody was raised against a 20 amino acid peptide identical to the C-terminus of rat ACIII, which is PAAFPNGSSVTLPHQVVDNP. A cysteine residue was added to the N-terminus to allow coupling to MBS-activated keyhole limpet hemocyanin. The antibody works on human cells also, as the corresponding peptide in the human AC3 is the peptide LATFPNGPSVTLPHQVVDNS, which differs at only three amino acids. The HGNC name for this protein is ADCY3.

Left: Western blots of rat olfactory epithelium (OE) and frontal cortex (FC). Our antibody stains bands at about 200kDa in olfactory epithelium which is rich in cilia. Fewer cilia are found in frontal cortex, and the protein is less heavily glycosylated, and a less prominent band is seen at about 160kDa. Data generated in the laboratory of Matt Sarkisian in the University of Florida. Right: Mixed neuron-glial cultures stained with our rabbit polyclonal antibody to ACIII (red) and our mouse monoclonal antibody to αII-spectrin (MCA-3D7 green). Note the strong and clean staining of neuronal cilia. Since αII-spectrin is specific for neurons in the CNS, the glial cells in this culture are not recognized by this antibody. The αII-spectrin antibody is also an excellent marker of neuronal plasma membranes.

Antibody Characteristics: The antibody was raised against the peptide PAAFPNGSSVTLPHQVVDNP with a cysteine residue added to the N-terminus to allow coupling to Keyhole Limpet Hemocyanin. The peptide corresponds the extreme C-terminus of the rat molecule. The antibody is provided in the form of affinity purified antibody at a concentration of 1mg/ml. Store at 4°C or -20°C. Avoid repeat freezing and thawing.

Suggestions for use: The antibody can be diluted to 1:500-1,000 for immunofluorescence staining and 1:5,000-10,000 for western blotting. On western blots look for a major bands at 160-200kDa, depending on the tissue.

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References:

1. Fuchs JL, Schwark HD. Neuronal primary cilia: a review. Cell Biol Int. 28:111-8 (2004).

2. Louvi A and Grove EA. Cilia in the CNS: the quiet organelle claims center stage. Neuron 69:1046-1060 (2011).

Price and Availability: - We currently supply 100μl aliquots of 1mg/ml purified antibody for $200. Material is in stock and ready for immediate shipping.

Limitations: This product is for research use only and is not approved for use in humans or in clinical diagnosis.

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