Journal
SPRINGERPLUS
Volume 5, Issue -, Pages -Publisher
SPRINGER INTERNATIONAL PUBLISHING AG
DOI: 10.1186/s40064-016-3330-y
Keywords
Rab3A; Fusion expression; Polyclonal antibody; Gel protein recovery; Protein interaction
Categories
Funding
- National Natural Science Foundation of China [31271135, 31070700]
- Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province [20134486]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available