GPNMB Antibody / Transmembrane glycoprotein NMB / HGFN (FY13098)

Immunohistochemical staining of GPNMB using anti-GPNMB antibody. GPNMB was detected in paraffin-embedded section of human liver cancer. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-GPNMB antibody overnight at 4oC. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Western blot analysis of GPNMB using anti-GPNMB antibody. The sample well of each lane was loaded with 30ug of sample under reducing conditions. Lane 1: rat liver tissue lysates, Lane 2: mouse liver tissue lysates, Lane 3: mouse kidney tissue lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GPNMB antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. The expected molecular weight of unglycosylated GPNMB is at ~64 kDa.

Immunohistochemical staining of GPNMB using anti-GPNMB antibody. GPNMB was detected in paraffin-embedded section of mouse brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-GPNMB antibody overnight at 4oC. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Immunofluorescent staining of GPNMB using anti-GPNMB antibody (green). GPNMB was detected in immunocytochemical section of cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2ug/ml rabbit anti-GPNMB antibody overnight at 4oC. DyLight 488 conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of U87 cells using anti-GPNMB antibody. Overlay histogram showing U87 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-GPNMB antibody (1ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1ug/million) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of HEPA1-6 cells using anti-GPNMB antibody. Overlay histogram showing HEPA1-6 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-GPNMB antibody (1ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1ug/million) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.