Polyclonal and monoclonal antibodies are widely used tools to localize, purify and study scarce proteins, including membrane proteins involved in neurotransmission. Antibodies of known epitope specificity (i.e. for which the sequence region of the protein forming the antibody epitope is known) are particularly useful tools to study structure and function of proteins involved in the function of biological transmitters and their receptors. Because the aminoacid sequences of many neurotransmitter receptors and ion channels are known, this information can be used to make synthetic peptides, and produce sequence specific antibodies. The proposed Core Facility will produce sequence-specific polyclonal antibodies for proteins of known sequence investigated by the members of the Center. If the use of sequence-specific monoclonal antibodies will be necessary, the facility will provide the services connected with immunization of the animals, testing of the sera to assess the optimal time for harvesting of their spleen B-cells, and establishment of hybridomas. The Facility will synthesize peptides corresponding to different sequence regions of the proteins under study, produce and characterize the polyclonal antibodies, and establish antipeptide hybridomas. When the goal will be to obtain an anti-peptide antibody able to cross-react with the native protein, the sequence regions for peptide synthesis will be selected after analysis of the protein sequence for maximum likelihood to be exposed on the surface of the protein. Short peptides will by produced by manual parallel synthesis in """"""""tea bags"""""""", longer peptides by automated synthesis in a peptide synthesizer. Polyclonal sera will be obtained by immunization of rabbits and chickens with i) peptides conjugated to a large protein carrier, or ii) linear peptides containing both the desired sequence region, and a second sequence segment forming a known strong T helper epitope, or iii) branched peptide oligomers, directly synthesized onto a branched lysine core. Monoclonal antibodies will be obtained from mice, and possibly from rabbits, after immunizations with the immunogens described above at ii) and iii). The anti-peptide antibody titer will be measured by ELISA and dot-blot assay. Cross-reactivity with the native protein will be assessed by ELISA or immunoprecipitation, using purified or semi-purified protein, provided by the individual interested investigators.
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