title: [Expression and purification of human recombinant LC3 in E.coli and preparation of its polyclonal antibodies].
abstract: OBJECTIVE
  To prepare the polyclonal antibodies against human LC3 protein and to provide a useful tool for monitoring cell autophagy.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Peking University, Beijing, China. HUL@novonordisk.com
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.forename: Jia
authors.initials: J
authors.lastname: Hu
authors.forename: Yan-jun
authors.initials: YJ
authors.lastname: Li
authors.forename: Huan
authors.initials: H
authors.lastname: Pan
authors.forename: Ying-yu
authors.initials: YY
authors.lastname: Chen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001343:Autophagy; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000906:Antibodies; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins