Northern Kentucky University

Characterization of Antibodies Raised Against Synthetic Peptides of a Novel P-type ATPase

Institution

Northern Kentucky University

Abstract

A putative magnesium-transporting P-Type ATPase was identified in a previous study. In order to gain further insights into the function of this novel transporter, antibodies were raised against selected peptide sequences of the protein. To identify immunogenic peptides the novel P-type ATPase was analyzed using the algorhythms of Hopp and Woods (predicts hydrophilic domains), Kyte and Doolittle (predicts hydrophobic domains), and Chou and Fasman (predicts B-turns). These programs predict which regions of a protein are hydrophilic and that tend to form loop structures such as β-turns, both of which are common at protein surfaces, and are a hallmark of antigenic peptides. Candidate peptides were then analyzed for glycosylation motifs because carbohydrates can shield antigenic sites. Two peptide sequences which met the aforementioned criteria were submitted to Sigma Genosys, a company that provides a complete service for polyclonal antibody production including peptide synthesis, conjugation, immunization and sera collection. ELISA assays were subsequently performed to determine the titer of the resultant immune sera. Peptide specific antibodies were then affinity purified from high titer immune sera using a sulfolink column. To accomplish this, the peptides were linked to the column material via a thioether bond. Immune sera were then passed over the columns, and following several wash steps, peptide specific antibodies were eluted. The highly purified antibodies will be used in Western blotting and immunohistochemistry experiments to determine the location and relative abundance of the novel P-type ATPase in tissues and cells.

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Characterization of Antibodies Raised Against Synthetic Peptides of a Novel P-type ATPase

A putative magnesium-transporting P-Type ATPase was identified in a previous study. In order to gain further insights into the function of this novel transporter, antibodies were raised against selected peptide sequences of the protein. To identify immunogenic peptides the novel P-type ATPase was analyzed using the algorhythms of Hopp and Woods (predicts hydrophilic domains), Kyte and Doolittle (predicts hydrophobic domains), and Chou and Fasman (predicts B-turns). These programs predict which regions of a protein are hydrophilic and that tend to form loop structures such as β-turns, both of which are common at protein surfaces, and are a hallmark of antigenic peptides. Candidate peptides were then analyzed for glycosylation motifs because carbohydrates can shield antigenic sites. Two peptide sequences which met the aforementioned criteria were submitted to Sigma Genosys, a company that provides a complete service for polyclonal antibody production including peptide synthesis, conjugation, immunization and sera collection. ELISA assays were subsequently performed to determine the titer of the resultant immune sera. Peptide specific antibodies were then affinity purified from high titer immune sera using a sulfolink column. To accomplish this, the peptides were linked to the column material via a thioether bond. Immune sera were then passed over the columns, and following several wash steps, peptide specific antibodies were eluted. The highly purified antibodies will be used in Western blotting and immunohistochemistry experiments to determine the location and relative abundance of the novel P-type ATPase in tissues and cells.