Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis

Background Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear. Results In this report the Rab3A gene from Rattus norvegicus was cloned and heterologously exp...
Ausführliche Beschreibung

Background Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear. Results In this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared. Conclusions The present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies. Ausführliche Beschreibung