Preparation and Application of Monoclonal Antibody Against hNDRG2

Abstract

The full-length hNdrg2 cDNA-coded 357 amino acids was cloned and expressed in Escherichia coli strain DH5alpha as a 6x His-tagged protein. The purified 6x His-fusion protein was used to immunize mice for preparing monoclonal antibodies (mAb) against N-myc downstream-regulated gene 2 (Ndrg2). A hybridoma secreting a monoclonal antibody against Ndrg2 was obtained and named FMU-Ndrg2.3. Western blot analysis confirmed that this mAb is specific only to Ndrg2 but not to Ndrg1, Ndrg3, and Ndrg4-B. Some tissue distribution features of Ndrg2 proteins, such as thyroid, kidney, testis, prostate, and pancreas islets, were present by immunohistochemistry.

NDRG2 is highly expressed in pancreatic β cells and involved in protection against lipotoxicity

“…Western blotting analysis confirmed that this antibody is specific only to NDRG2 and not to NDRG1, NDRG3, and NDRG4 protein [18]. Human and mouse pancreatic tissues were fixed in a freshly prepared solution of 4% paraformaldehyde, dehydrated through graded solutions of ethanol, and embedded in paraffin.…”

Expression and prognostic value of NDRG2 in human astrocytomas

“…After washing with PBS for 3 times, nonspecific binding was blocked by incubating sections with 10% normal goat serum in PBS for 1 h at room temperature. Without washing, the sections were incubated with mouse anti-human NDRG2 (1:100) [9] in PBS containing 1% BSA and 0.3% Trition-X100 at 4°C overnight. After washing in PBS, the sections were incubated with biotinylated goat anti-mouse IgG (1:400, Vector Laboratories) for 1 h at room temperature, and then detected with a streptavidin-peroxidase complex (1:200, Jackson ImmunoResearch).…”

Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development

“…The relative level of NDRG2 expression in rat testis was evaluated by Western blot assay, as described previously (Hu et al 2006;Liu et al 2009). Briefly, the tissue or cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto a 0.2-μm nitrocellulose membrane, followed by a blocking step with 5% non-fat milk in TRIS-buffered saline with Tween-20 for 1 h at 37°C.…”

“…The expression of NDRG2 was characterized by immunohistochemistry with anti-NDRG2, FMU-NDRG cl2.3 (Hu et al 2006;Liu et al 2009). Briefly, the sections (4 μm) were dewaxed in xylene, dehydrated through descending concentrations of ethanol, immersed in 0.3% H 2 O 2 -methanol for 30 min, washed with PBS, probed with FMU-NDRG cl2.3 (1:100) at 4°C overnight in a moist box, washed, and incubated with biotinylated goat anti-mouse IgG (1:300) at room temperature for 2 h. Immunostaining was detected with the streptavidinperoxidase complex and 0.1% diaminobenzidine (DAB), 0.05% H 2 O 2 in PBS (giving a brown color).…”