Resumo

During the nervous system development, the neuron has to navigate through many diverse substrates constituted of different cells, extracellular matrix molecules and neuron processes. Such navigation is not only observed during the embryogenesis but also in pathologic processes and re-growth in the nervous system, guided through a variety of protein interactions. There are many mechanisms and molecules that guide axon growth to specific targets, and these molecules are present in the developmental stage and also on regeneration stages of the nervous system. Among these molecules, Semaphorins (SEMAs) constitute a large secreted or membrane-associated protein family, many proteins of which play a roll on axon navigation, fasciculation, ramification and synapse formation, acting like chemorepellents or chemoattractive molecules. The SEMA family has at least twenty different members in vertebrates and three members in invertebrates. Of all the SEMAs, little is know about the class 5 SEMAs, especially about Sema 5B. The few papers published about class 5 SEMAs elucidate some aspects of Sema 5A, but in relation to Sema 5B less literature is found. Our work was to obtain anti-bodies á- Mouse Sema 5B, for posterior applications as tools for studying Sema 5B. To obtain these, the recombinant cytoplasmic domain of Semaphorin 5B was synthesized in DH5á in fusion with His tag domain (His6-CITO) and purified using nickel agarose chromatography column (Ni-NTA). The recombinant domain was submitted to SDS-PAGE and used to immunize rabbits and mice. The resulting polyclonal sera were tested using Western Blot technique. The rabbit serum wasn’t capable to identify the recombinant protein. Nevertheless, three mouse sera were able to recognize the His6-Cito Sema 5B protein on 1:400 through to 1:12800 dilutions. Afterwards the sera were tested with endogenous Sema 5B obtained from mouse brain extract, with positive results.