Anti-Doublecortin Antibody

Doublecortin is a microtubule-associated protein found in neuroblasts and immature neurons, especially those undergoing embryonic migrations. It was first identified as the X-chromosome-encoded gene product mutated in the human diseases Lissencephaly (in males) and Periventricular Heterotopia (in females), diseases caused by failure of neocortical neuroblast migration. The human protein itself is 360 amino acids (40,044 daltons) and contains doublecortin motifs near its N-terminus that are believed to mediate the microtubule binding.

Store at 4°C in the dark. Under these conditions, the antibodies should have a shelf life of at least twelve months, provided they remain sterile. For longer term storage, aliquot and freeze to avoid freeze-thaw of the antibody.

Chickens were immunized with two synthetic peptide / keyhole limpet hemocyanin (KLH) conjugates. These synthetic peptides corresponded to different regions of the doublecortin gene product, but are shared between the human (CAA06617.1, NCBI) and mouse (AAT58219.1, NCBI) sequences. After repeated injections, immune eggs were collected, and the IgY fractions were purified from the yolks. These IgY fractions were then affinity purified using a peptide column, and the concentrations of the eluates adjusted to 200 μg/mL. Finally, equal volumes of these affinity-purified anti-peptide antibodies were mixed, and the preparation was filter-sterilized.

This anti-peptide antibody mixture was analyzed by immunohistochemistry (at a dilution of 1:2000) using fluorescein-labeled goat anti-chicken IgY (1:500 dilution, Aves Labs Cat.# F-1005) as the secondary reagent.