title: [Preparation and characterization of a polyclonal antibody against human Fibrocystin-L].
abstract: OBJECTIVE
  PKHDL1 (the gene for Polycystic Kidney and Hepatic Disease Like-1) had been recently identified, but characteristics of the gene product, Fibrocystin-L (FPC-L), still remain unknown. We therefore produced a rabbit polyclonal antibody hFL-Np to explore the cellular characteristics of this novel protein.
  
  
  METHODS
  Based on the hydrophobic/hydrophilic analyses, chose a cDNA fragment which encodes 633L-768K amino acids of the FPC-L and amplified it by RT-PCR. The PCR product was then cloned into a prokaryotic expression vector pGEX-GST. With IPTG induction, the antigen hFL-N was produced and further purified. A rabbit was immunized with the antigen and its antiserum was collected. Applied Western blot with the polyclonal antiserum hFL-Np and validated the antibody specific for FPC-L protein. In addition, also used immunofluorescence staining with hFL-Np to detect the subcellular distribution in cultured HEK293 cells.
  
  
  RESULTS
  The prokaryotic expression vector pGEX-hFL-N was successfully constructed and a hFL-N antigen was produced in E.coli Rossetta cells. Using the antigen, a polyclonal antibody hFL-Np was produced and the specificity for FPC-L was also proved by biochemistry and cellular assays. Using the antibody, the cellular staining reveals that FPC-L was a cytosolic protein.
  
  
  CONCLUSIONS
  We produced an anti-FPC-L polyclonal antibody hFL-Np. By biochemistry and cellular characterization, proved that the polyclonal antibody hFL-Np is specific for FPC-L and demonstrated FPC-L is a cytosolic protein. The finding provides a platform for further dissecting FPC-L functions in mammalian development.
authors.affiliation: Divosion of Translational Cancer Research and Therapy, State Key Laboratory of Molecular Oncology, Cancer Hospital and Institute, Chinese Academy of Medical Sciences, Beijing 100021, China. lianpeiwen@yahoo.com.cn
authors.forename: Pei-wen
authors.initials: PW
authors.lastname: Lian
authors.forename: Yu-long
authors.initials: YL
authors.lastname: Fu
authors.forename: Ao
authors.initials: A
authors.lastname: Li
authors.forename: Bao-zhen
authors.initials: BZ
authors.lastname: Dai
authors.forename: Zhen-wei
authors.initials: ZW
authors.lastname: Ding
authors.forename: Lan
authors.initials: L
authors.lastname: Li
authors.forename: Guan-qing
authors.initials: GQ
authors.lastname: Wu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D000906:Antibodies; C478647:PKHD1L1 protein, human; D011956:Receptors, Cell Surface