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.