Preparation of rabbit anti-rat LRRN3 polyclonal antibody and study of its expression

BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.