title: A quantitative analysis of N-myc downstream regulated gene 2 (NDRG 2) in human tissues and cell lysates by reverse-phase protein microarray.
abstract: BACKGROUND
  N-myc downstream regulated gene 2 (NDRG2) belongs to the NDRG family, which is comprised of 4 members, NDRG1-4. Recently, NDRG2 was reported as a new candidate for a tumor suppressor gene. We developed a reverse-phase protein microarray assay to access NDRG2 levels in human tissue specimens and cell lines.
  
  
  METHODS
  We synthesized recombinant NDRG2 protein and produced monoclonal antibodies (mAb) to the NDRG2 protein. We selected 2 hybridomas producing mAb that specifically recognize the NDRG2 protein. To determine the NDRG2 concentration, the samples of serially-diluted NDRG2 protein, cell lysate, or tissue lysate were spotted onto a nitrocellulose membrane-coated slide glass and allowed to react with the mAb to the NDRG2 protein. The reaction was followed by additional incubation with biotin-linked anti-mouse IgG and horseradish peroxidase (HRP)-conjugated streptavidin, subsequently. The addition of dimethylaminobenzidine induced color development, which was measured using the GenePix program. We determined the NDRG2 concentration in various tissue specimens and cell lines using the new protein microarray technique.
  
  
  RESULTS
  The dose-response relationship between NDRG2 and color intensity showed linearity in a range 0-10 ng/ml and a sensitivity of 50 pg/ml. The NDRG2 concentrations in the liver tissue lysates of patients with hepatocellular carcinoma (52.0+21.5 ng/mg) were significantly diminished as compared with those in the normal liver tissues (549.6+94.6 ng/mg). The results of the assay showed good agreement with those of Western blot analysis.
  
  
  CONCLUSIONS
  The protein microarray is a highly sensitive and accurate method, and can adopted to assess specific proteins in human tissues or cell lines, particularly in the field of cancer and pathological research.
authors.affiliation: Stem Cell Research Center, KRIBB, Daejeon 305-806, South Korea.
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Park
authors.forename: Seung-Chul
authors.initials: SC
authors.lastname: Choi
authors.forename: Hayang Sin
authors.initials: HS
authors.lastname: Lee
authors.forename: Daekwang
authors.initials: D
authors.lastname: Kim
authors.forename: Kyoung Eun
authors.initials: KE
authors.lastname: Baek
authors.forename: Jong Tae
authors.initials: JT
authors.lastname: Kim
authors.forename: Jong-Seok
authors.initials: JS
authors.lastname: Lim
authors.forename: Young Il
authors.initials: YI
authors.lastname: Yeom
authors.forename: Jin Woong
authors.initials: JW
authors.lastname: Chung
authors.forename: Jong Wan
authors.initials: JW
authors.lastname: Kim
authors.forename: Pyung-Keun
authors.initials: PK
authors.lastname: Myung
authors.forename: Hee Gu
authors.initials: HG
authors.lastname: Lee
authors.forename: Jae Wha
authors.initials: JW
authors.lastname: Kim
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Song
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; C466228:NDRG2 protein, human; D025521:Tumor Suppressor Proteins