SPECIFICITY OF ANTIBODIES: UNEXPECTED CROSS-REACTIVITY OF ANTIBODIES DIRECTED AGAINST THE EXCITATORY AMINO ACID TRANSPORTER 3 (EAAT3)

摘要: Abstract Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of is often straightforward, determination the antibody specificity not. Here we illustrate this by describing production and characterization to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding parts EAAT3 sequence immunized 6 sheep 30 rabbits. All sera were affinity purified against relevant immobilized peptide. Antibodies obtained almost all cases. Immunoblotting with tissue extracts from wild type knockout animals revealed that most did not recognize native protein, some recognized other proteins. Several immunization protocols tried, but strong reactions only seen C-terminal peptides. In contrast, good several EAAT2. was detected neurons. However, rabbits an EAAT3-peptide residues 479–498 produced labeled axoplasm microtubules therein particularly strongly. On blots, these both a slightly smaller, far more abundant protein turned out be tubulin. The fractionated on columns One fraction contained apparently specific while another recognizing tubulin despite lack primary identity between two Addition free peptide incubation solution blocked immunostaining Conclusions: Not synthetic protein. important than protocol. hard predict because cross-reactivity can unrelated molecules. antigen preabsorption test little value testing antibodies.