PMID: 22558991May 9, 2012Paper

[Expression and purification of human recombinant LC3 in E.coli and preparation of its polyclonal antibodies].

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
Hua LiYing-yu Chen

Abstract

To prepare the polyclonal antibodies against human LC3 protein and to provide a useful tool for monitoring cell autophagy. The plasmid of pET32a(+)LC3 was successfully constructed, human LC3 protein was purified from E.coli through one-step purification with SP-Sepharose XL. The purified LC3 protein was used to immunize rabbits for preparing the specific antibody. Simultaneously, it was also coupled with CNBr-activated Sepharose 4B. The specificity of the polyclonal anti-LC3 antibody was determined by ELISA and Western blotting. Recombinant human LC3 protein was expressed and purified successfully. Prepared polyclonal anti-LC3 antibody showed high specificity against LC3 without cross-reaction with E.coli components in Western blotting. It was also indicated that the antibody could recognize both LC3-I and LC3-II in the process of cell autophagy. Human recombinant LC3 protein successfully expressed in E.coli and purified. Polyclonal anti-LC3 antibody with high specificity is prepared and applied successfully in detection of cell autophagy.

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