Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human

Cited:

Human, Mouse, Rat, Xenograft

Applications

Validated:

Immunohistochemistry, Western Blot, ELISA Capture (Matched Antibody Pair), Immunoprecipitation

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Flow Cytometry, Immunocytochemistry, FACS

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all Osteoactivin/GPNMB Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human Osteoactivin isoform b
Lys23-Asn486
Accession # NP_002501

Specificity

Detects human Osteoactivin in ELISAs and Western blots. In sandwich immunoassays, less than 2% cross-reactivity with recombinant mouse Osteoactivin is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human Osteoactivin/GPNMB Antibody

Detection of Human Osteoactivin/GPNMB antibody by Western Blot.

Detection of Human Osteoactivin/GPNMB by Western Blot.

Western blot shows lysates of U-118-MG human glioblastoma/astrocytoma cell line and T98G human glioblastoma cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human Osteoactivin/GPNMB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2550) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). Specific bands were detected for Osteoactivin/GPNMB at approximately 95 and 120 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Osteoactivin/GPNMB antibody in Human Liver by Immunohistochemistry (IHC-P).

Osteoactivin/GPNMB in Human Liver.

Osteoactivin/GPNMB was detected in immersion fixed paraffin-embedded sections of human liver using Goat Anti-Human Osteoactivin/GPNMB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2550) at 3 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to Kupffer cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Osteoactivin/GPNMB Specificity is Shown by Immunocytochemistry in Knockdown Cell Line.

Osteoactivin/GPNMB Specificity is Shown by Immunocytochemistry in Knockdown Cell Line.

U‑87 MG human glioblastoma/astrocytoma parental cell line ctrl and Osteoactivin/GPNMB U-87 MG KD cells were labelled with a green or a far-red fluorescent dye, respectively. Cells were stained with Goat Anti-Human Osteoactivin/GPNMB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2550) followed by incubation with an Alexa-fluor 555 conjugated secondary antibody (upper panel). DAPI-only counterstained cells shown on a lower panel. Acquisition of the blue (nucleus-DAPI), green (identification of ctrl cells), red (antibody staining) and far-red (identification of KD cells) channels was performed. Representative images of the blue and red (grayscale) channels are shown. Ctrl and KD cells are outlined with green and magenta dashed line, respectively. Primary antibody concentration used: 0.2 µg/mL. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).
Western Blot Shows Osteoactivin/GPNMB Specificity Using Knockdown Cell Line.

Western Blot Shows Osteoactivin/GPNMB Specificity Using Knockdown Cell Line.

Western blot shows lysates of U‑87 MG human glioblastoma/astrocytoma parental cell line and Osteoactivin/GPNMB knockdown U-87 MG cell line (KD). Nitrocellulose membrane was probed with 0.4 µg/mL Goat Anti-Human Osteoactivin/GPNMB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2550) followed by HRP-conjugated secondary antibody. Specific bands were detected for Osteoactivin/GPNMB at approximately 76 and 120 kDa (as indicated) in the parental U-87 MG cell line, but is significantly reduced in the U-87 MG cell line. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.
Detection of Osteoactivin/GPNMB by Immunoprecipitation.

Detection of Osteoactivin/GPNMB by Immunoprecipitation.

U‑87 MG human glioblastoma/astrocytoma cell line lysates were prepared and immunoprecipitation was performed using 2.0 μg of Goat Anti-Human Osteoactivin/GPNMB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2550) pre-coupled to Dynabeads Protein G. Immunoprecipitated Osteoactivin/GPNMB was detected in Western Blot with a Rabbit Osteoactivin/GPNMB Antibody. The Ponceau stained transfer of the blot is shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; HC=antibody heavy chain. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).
Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

GPNMB and galectin-3 levels are elevated in FTD-GRN brains. a, b GPNMB and galectin-3 levels (ng/mg protein) were measured in frontal lobe tissue lysates generated from cognitively normal controls (CTL; n = 27) and FTD-GRN patients (n = 25). Data analyzed using unpaired t-test. c Representative immunoblots for GPNMB and galectin-3 in frontal lobe lysates from cognitively normal controls (n = 8) and FTD-GRN (n = 8) patients. d GPNMB levels (ng/mL) in CSF samples form cognitively normal controls (n = 14), FTD-GRN (n = 9), FTD-C9orf72 (n = 12) and FTD-MAPT (n = 12) samples quantified by ELISA. Data analyzed using one-way ANOVA. e, f GPNMB immunostaining was performed on frontal lobe tissue sections from cognitively normal controls (n = 5) (e) and FTD-GRN (n = 5) (f) patients. g, h Immunostaining for p-TDP 43 was stained on adjacent sections from identical samples in e, f as marker of FTLD pathology. i GPNMB staining intensity in human brain sections (e, f) were measured and presented as fold change. Representative immunofluorescence staining for cell markers (green) (j, n, r), GPNMB (red) (k, o, s), DAPI (blue) (i, p, t) in paraffin sections of brains from FTD-GRN cases. Iba-1, GFAP, NeuN used for markers of human microglia, astrocytes, and neurons respectively. GPNMB and Iba-1 signals overlap (arrow) (m) whereas, no overlapping signal was observed in co-staining with GFAP or NeuN (q, u). Scale bars were labeled in the images. Data analyzed by unpaired t-test. Scale bars (20 µm) labeled in images and quantitative data are shown as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33028409), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

GPNMB and galectin-3 levels are elevated in FTD-GRN brains. a, b GPNMB and galectin-3 levels (ng/mg protein) were measured in frontal lobe tissue lysates generated from cognitively normal controls (CTL; n = 27) and FTD-GRN patients (n = 25). Data analyzed using unpaired t-test. c Representative immunoblots for GPNMB and galectin-3 in frontal lobe lysates from cognitively normal controls (n = 8) and FTD-GRN (n = 8) patients. d GPNMB levels (ng/mL) in CSF samples form cognitively normal controls (n = 14), FTD-GRN (n = 9), FTD-C9orf72 (n = 12) and FTD-MAPT (n = 12) samples quantified by ELISA. Data analyzed using one-way ANOVA. e, f GPNMB immunostaining was performed on frontal lobe tissue sections from cognitively normal controls (n = 5) (e) and FTD-GRN (n = 5) (f) patients. g, h Immunostaining for p-TDP 43 was stained on adjacent sections from identical samples in e, f as marker of FTLD pathology. i GPNMB staining intensity in human brain sections (e, f) were measured and presented as fold change. Representative immunofluorescence staining for cell markers (green) (j, n, r), GPNMB (red) (k, o, s), DAPI (blue) (i, p, t) in paraffin sections of brains from FTD-GRN cases. Iba-1, GFAP, NeuN used for markers of human microglia, astrocytes, and neurons respectively. GPNMB and Iba-1 signals overlap (arrow) (m) whereas, no overlapping signal was observed in co-staining with GFAP or NeuN (q, u). Scale bars were labeled in the images. Data analyzed by unpaired t-test. Scale bars (20 µm) labeled in images and quantitative data are shown as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33028409), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

Detection of Human Osteoactivin/GPNMB by Immunohistochemistry

GPNMB and galectin-3 levels are elevated in FTD-GRN brains. a, b GPNMB and galectin-3 levels (ng/mg protein) were measured in frontal lobe tissue lysates generated from cognitively normal controls (CTL; n = 27) and FTD-GRN patients (n = 25). Data analyzed using unpaired t-test. c Representative immunoblots for GPNMB and galectin-3 in frontal lobe lysates from cognitively normal controls (n = 8) and FTD-GRN (n = 8) patients. d GPNMB levels (ng/mL) in CSF samples form cognitively normal controls (n = 14), FTD-GRN (n = 9), FTD-C9orf72 (n = 12) and FTD-MAPT (n = 12) samples quantified by ELISA. Data analyzed using one-way ANOVA. e, f GPNMB immunostaining was performed on frontal lobe tissue sections from cognitively normal controls (n = 5) (e) and FTD-GRN (n = 5) (f) patients. g, h Immunostaining for p-TDP 43 was stained on adjacent sections from identical samples in e, f as marker of FTLD pathology. i GPNMB staining intensity in human brain sections (e, f) were measured and presented as fold change. Representative immunofluorescence staining for cell markers (green) (j, n, r), GPNMB (red) (k, o, s), DAPI (blue) (i, p, t) in paraffin sections of brains from FTD-GRN cases. Iba-1, GFAP, NeuN used for markers of human microglia, astrocytes, and neurons respectively. GPNMB and Iba-1 signals overlap (arrow) (m) whereas, no overlapping signal was observed in co-staining with GFAP or NeuN (q, u). Scale bars were labeled in the images. Data analyzed by unpaired t-test. Scale bars (20 µm) labeled in images and quantitative data are shown as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33028409), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Osteoactivin/GPNMB by Western Blot

Detection of Osteoactivin/GPNMB by Western Blot

Dasatinib can upregulate the expression of gpNMB in MDA-MB-468-bearing mice. The mice were separated into two groups and were treated with either dasatinib or vehicle control for 21 days. Subgroups of mice (n = 4) were euthanized at different timepoints. Tumors were then subjected to Western blot analyses of gpNMB and ꞵ-actin. (A) Western blot images of gpNMB and ꞵ-actin; (B) Plot of normalized gpNMB expression with ꞵ-actin. DAS = dasatinib. ns = not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36900378), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Osteoactivin/GPNMB by Western Blot

Detection of Osteoactivin/GPNMB by Western Blot

Dasatinib can upregulate the expression of gpNMB on MDA-MB-468 and MDA-MB-231 cells in vitro. (A) Western blot for gpNMB, ꞵ-actin, and dasatinib-related proteins p-Src, Src; (B) Normalized gpNMB expression relative to ꞵ-actin. DAS = dasatinib. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36900378), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Osteoactivin/GPNMB by Western Blot

Detection of Osteoactivin/GPNMB by Western Blot

Dasatinib can upregulate the expression of gpNMB on MDA-MB-468 and MDA-MB-231 cells in vitro. (A) Western blot for gpNMB, ꞵ-actin, and dasatinib-related proteins p-Src, Src; (B) Normalized gpNMB expression relative to ꞵ-actin. DAS = dasatinib. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36900378), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Osteoactivin/GPNMB by Western Blot

Detection of Osteoactivin/GPNMB by Western Blot

Dasatinib can upregulate the expression of gpNMB in MDA-MB-468-bearing mice. The mice were separated into two groups and were treated with either dasatinib or vehicle control for 21 days. Subgroups of mice (n = 4) were euthanized at different timepoints. Tumors were then subjected to Western blot analyses of gpNMB and ꞵ-actin. (A) Western blot images of gpNMB and ꞵ-actin; (B) Plot of normalized gpNMB expression with ꞵ-actin. DAS = dasatinib. ns = not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36900378), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Osteoactivin/GPNMB Antibody

Application
Recommended Usage

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human liver

Immunoprecipitation

2 µg/1 mg cell lysate
Sample: Cell lysate of U-87 MG human glioblastoma/astrocytoma cell line

Western Blot

0.5 µg/mL
Sample: U‑118‑MG human glioblastoma/astrocytoma cell line and T98G human glioblastoma cell line

Human Osteoactivin/GPNMB Sandwich Immunoassay

ELISA Capture (Matched Antibody Pair)
Recommended Concentration: 0.2-0.8 µg/mL
Use in combination with these reagents:
  • Detection Reagent: Human Osteoactivin/GPNMB Biotinylated Antibody (Catalog # BAF2550)
  • Standard: Recombinant Human Osteoactivin/GPNMB Fc Chimera Protein, CF (Catalog # 2550-AC)
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 3 reviews rated 5 using AF2550 in the following applications:

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. See Certificate of Analysis for details.
*Small pack size (-SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Osteoactivin/GPNMB

Osteoactivin (also GPNMB and DC-HIL) is a variably glycosylated 75 - 125 kDa member of the NMB/pMEL-17 family of molecules. It is found in multiple subcellular sites, but is most often associated with the endosomal/lysosomal compartment (1-3). Human Osteoactivin is a 560 amino acid (aa) type I transmembrane protein. Its precursor contains a 21 aa signal sequence, a 465 aa luminal/extracellular domain, a 21 aa transmembrane segment and a 53 aa cytoplasmic tail (4, 5). The luminal region contains an N-terminal heparin-binding motif (aa 23-26), multiple glycosylation sites, an RGD motif (aa 64-66) and an 88 aa PKD domain (aa 240-327). The intracellular tail has an ITAM (Y-x-x-I) and lysosomal targeting (L-L) motif (4, 5). The extracellular/luminal region shares 74% and 77% aa identity with the equivalent regions in mouse and canine, respectively. Multiple isoforms would appear to exist. There is one alternate splice form known that shows a 12 aa insert between aa 339-340 (6). An addtional 206 aa isoform shows a mutation at position # 181 that results in a 26 aa substitution for the C-terminal 380 amino acids (7, 8). This has the potential to be soluble and may represent a counterpart to a secreted isoform of rat Osteoactivin (9). Cells known to express Osteoactivin include macrophages/Kupffer cells, fibroblasts, osteoblasts, myeloid dendritic cells, retinal pigment epithelial cells and melanocytes, plus fetal chondrocytes and stratum basale keratinocytes (3-5, 10-12). In mice, Osteoactivin is reported to bind to heparan sulfate-proteoglycan, possibly on the surface of endothelial cells and may also interact with integrins (13). It also appears to act as an inflammatory suppressor gene, as its expression downregulates the macrophage inflammatory response by inhibiting IL-6 and IL-12 p40 production (3).

References

  1. Bachner, D. et al. (2002) Gene Exp. Patterns 1:159.
  2. Safadi, F.F. et al. (2002) J. Cell. Biochem. 84:12.
  3. Ripoll, V.M. et al. (2007) J. Immunol. 178:6557. 
  4. Owen, T.A. et al. (2003) Crit. Rev. Eukaryot. Gene Expr. 13:205. 
  5. Weterman, M.A.J. et al. (1995) Int. J. Cancer 60:73. 
  6. Kuan, C-T. et al. (2006) Clin. Cancer Res. 12:1970. 
  7. Lennerz, V. et al. (2005) Proc. Natl. Acad. Sci. USA 102:16013.
  8. Genbank Accession # AAH11595.
  9. Abdelmagid, S.M. et al. (2007) J. Cell. Physiol. 210:26.
  10. Haralanova-Ilieva, B. et al. (2005) J. Hepatol. 42:565.
  11. Ahn, J.H. et al. (2002) Blood 100:1742.
  12. Anderson, M.G. et al. (2002) Nat. Genet. 30:81.
  13. Shikano, S. et al. (2001) J. Biol. Chem. 276:8125.

Long Name

Glycoprotein Non-Metastatic Melanoma Protein B

Alternate Names

DC-HIL, GPNMB, HGFIN

Entrez Gene IDs

10457 (Human); 93695 (Mouse); 113955 (Rat); 102133517 (Cynomolgus Monkey)

Gene Symbol

GPNMB

UniProt

NP_002501

Additional Osteoactivin/GPNMB Products

Product Documents for Human Osteoactivin/GPNMB Antibody

Certificate of Analysis

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Product Specific Notices for Human Osteoactivin/GPNMB Antibody

For research use only

Citations for Human Osteoactivin/GPNMB Antibody

Customer Reviews for Human Osteoactivin/GPNMB Antibody (3)

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  • Human Osteoactivin/GPNMB Antibody
    Name: Laboratorio Inmuno-Oncología
    Application: Immunocytochemistry/Immunofluorescence
    Sample Tested: Melanoma tissue
    Species: Human
    Verified Customer | Posted 09/27/2023
    Human Osteoactivin/GPNMB Antibody AF2550
  • Human Osteoactivin/GPNMB Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Adult brain (hippocampus)
    Species: Human
    Verified Customer | Posted 02/23/2023
    primary Ab 1 :1000
    Human Osteoactivin/GPNMB Antibody AF2550
  • Human Osteoactivin/GPNMB Antibody
    Name: Anonymous
    Application: Immunohistochemistry
    Sample Tested: Adult spinal cord
    Species: Human
    Verified Customer | Posted 01/21/2021
    Human specimens were deparaffinized and rehydrated before immunohistochemistry. The sections were blocked with 10% goat serum in PBS, then incubated overnight at 4 °C with the GPNMB antibody.
    Human Osteoactivin/GPNMB Antibody AF2550

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