Pecificity of Antibodies: Unexpected Cross-reactivity of Ntibodies Directed against the Excitatory Amino Acid Ransporter 3 (eaat3)
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چکیده
bstract—Specific antibodies are essential tools for identifyng individual proteins in biological samples. While generaion of antibodies is often straightforward, determination of he antibody specificity is not. Here we illustrate this by escribing the production and characterization of antibodies o excitatory amino acid transporter 3 (EAAT3). We syntheized 13 peptides corresponding to parts of the EAAT3 seuence and immunized 6 sheep and 30 rabbits. All sera were ffinity purified against the relevant immobilized peptide. Anibodies to the peptides were obtained in almost all cases. mmunoblotting with tissue extracts from wild type and AAT3 knockout animals revealed that most of the antibodes did not recognize the native EAAT3 protein, and that some ecognized other proteins. Several immunization protocols ere tried, but strong reactions with EAAT3 were only seen ith antibodies to the C-terminal peptides. In contrast, good ntibodies were obtained to several parts of EAAT2. EAAT3 as only detected in neurons. However, rabbits immunized ith an EAAT3-peptide corresponding to residues 479–498 roduced antibodies that labeled axoplasm and microtubules herein particularly strongly. On blots, these antibodies recgnized both EAAT3 and a slightly smaller, but far more bundant protein that turned out to be tubulin. The antibodies ere fractionated on columns with immobilized tubulin. One raction contained antibodies apparently specific for EAAT3 hile another fraction contained antibodies recognizing both AAT3 and tubulin despite the lack of primary sequence dentity between the two proteins. Addition of free peptide to he incubation solution blocked immunostaining of both AAT3 and tubulin. Conclusions: Not all antibodies to syn-
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تاریخ انتشار 2005