Anti gpnmb

Manufactured by R&D Systems

Sourced in United States

Anti-GPNMB is a recombinant protein that can be used as a research tool. It represents the extracellular domain of the human GPNMB (Glycoprotein NMB) protein.

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Lab products found in correlation

Electroblotting apparatus, by Bio-Rad (2 mentions) Kaluza software, by Beckman Coulter (2 mentions) Pe conjugated anti cd68, by Bio-Rad (2 mentions) Anti tau, by Cell Signaling Technology (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Odyssey v3, by LI COR (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Irdye conjugated antibody, by LI COR (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Whatman, by Cytiva (1 mentions) Nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) Anti β catenin, by Santa Cruz Biotechnology (1 mentions) Imagequant software, by Cytiva (1 mentions) Lysis buffer, by Bioss Antibodies (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Anti mmp 9, by Merck Group (1 mentions) Anti mitf, by Merck Group (1 mentions) Radioimmunoprecipitation assay (ripa), by Santa Cruz Biotechnology (1 mentions) Anti hmb 45, by Agilent Technologies (1 mentions) Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

GPNMB (6 citations) Human Osteoactivin/GPNMB anti goat (4 citations) Goat anti-mouse Gpnmb (2 citations) Goat anti-human GPNMB (2 citations) Anti-Gpnmb antibody (2 citations) Goat anti-GPNMB antibody (2 citations) Goat anti-GPNMB (2 citations)

7 protocols using anti gpnmb

Immunoblotting Analysis of Brain and CSF

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Brain homogenates and CSF samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were incubated with the following primary antibodies: anti-GPNMB (R&D systems, catalog number AF2550), anti-albumin (Dako Cytomation, catalog number A0001), anti-Tau (Cell signaling, catalog number 4019S), anti-mouse P-Tau (Cell signaling, catalog number 9632S), anti-human P-tau (Santa Cruz, catalog number 101815) and anti-AT8 (Innogenetics, catalog number 90206), followed by a horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using the SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific).

Zigdon H., Savidor A., Levin Y., Meshcheriakova A., Schiffmann R, & Futerman A.H. (2015). Identification of a Biomarker in Cerebrospinal Fluid for Neuronopathic Forms of Gaucher Disease. PLoS ONE, 10(3), e0120194.

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Western Blot Protein Detection Protocol

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Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 50 mM Tris—HCl pH 7.4, 2 mM EDTA, 0.5% deoxycholate, 1mM Na3VO4, 20 mM NaF, 0.5%Triton X-100), supplemented with protease inhibitor cocktail (Roche Applied Science) and phenylmethylsulfonyl fluoride (PMSF). Lysates were cleared by centrifugation at 4°C for 10 min at 10,000 × g. Equal amounts of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies used were the polyclonal anti-Gpnmb (R&D Systems) and anti-tubulin-alpha (Cedarlane Laboratories Limited). Matching secondary IRDye-conjugated antibodies (Westburg) were used for detection in an Odyssey V3.0 (LI-COR Inc.).

Marques A.R., Gabriel T.L., Aten J., van Roomen C.P., Ottenhoff R., Claessen N., Alfonso P., Irún P., Giraldo P., Aerts J.M, & van Eijk M. (2016). Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease. PLoS ONE, 11(1), e0147208.

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Protein Expression Analysis Protocol

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Total proteins were obtained from cell lysates using lysis buffer (Bioss, China), and nuclear proteins were collected by Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, USA). Equal amounts of proteins were separated by SDS-PAGE, and electrophoretically transferred to a PVDF membrane. The membranes were incubated with primary antibodies (anti-GPNMB 1:2000 dilution, R&D Systems, USA; anti-MMP-2, MMP-9 1:1000 dilution, Abcam; anti-β-catenin 1:1000 dilution, Santa Cruz, USA; mouse anti-β-actin and LaminB1, 1:2000 dilution, Abcam, UK) at 4°C overnight, followed by treatment with secondary antibodies for 1 h at room temperature. Bands were then visualized by ECL (Amersham Pharmacia GE, China), and analyzed using Image Quant software (Amersham BioSciences, USA).

Xu S., Fan Y., Li D., Liu Y, & Chen X. (2018). Glycoprotein nonmetastatic melanoma protein B accelerates tumorigenesis of cervical cancer in vitro by regulating the Wnt/β-catenin pathway. Brazilian Journal of Medical and Biological Research, 52(1), e7567.

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Protein Expression Analysis in Uteri and ELT3 Cells

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Uteri and ELT3 cells were homogenized in RIPA (Santa Cruz, CA, USA). Western blots used 1:1000 rabbit polyclonal anti-phospho-S6 (Ser 235/236), anti-S6, anti-phospho-4EBP1 (Thr37/46), anti-4EBP1, anti-phospho-p44/42-ERK1/2 (T202/Y204), p44/42-ERK1/2, and 1:5000 anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Cell Signaling) antibodies. IHC was performed, as described in Prizant et al. (2013) (link). Sections were incubated with 1:1000 rabbit anti-SMA (Epitomics, Burlingame, CA, USA), 1:400 anti-phospho-S6 (Ser 235/236), 1:100 anti-MMP-9, 1:300 anti-MITF, 1:500 anti-GPNMB (Sigma-Aldrich, for mouse), 1:100 anti-GPNMB (R&D Systems, for human) (Hoashi et al. 2010 (link), Rose et al. 2010b), or 1:50 anti-HMB-45 (Dako) primary antibody. For immunofluorescence, sections were incubated with 1:200 fluorescein-conjugated anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Nuclei were labeled using 1 mg/mL Hoechst-33258 (Invitrogen). For immunofluorescence, cells were grown in poly-d-lysine pre-coated glass bottom dishes (MatTech Corporation, Ashland, MA, USA), fixed in 4% paraformaldehyde, and stained with 1:250 anti-MITF or 1:300 anti-GPNMB (Sigma-Aldrich) primary antibody.

Prizant H., Taya M., Lerman I., Light A., Sen A., Mitra S., Foster T.H, & Hammes S.R. (2016). Estrogen maintains myometrial tumors in a lymphangioleiomyomatosis model. Endocrine-related cancer, 23(4), 265-280.

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Phagocytic capacity of LMNCs

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LMNCs (5X10⁵ cells/200 μl) were incubated with 3 μl FITC-microspheres (1X10⁸) for 20 min [27 (link)]. Thereafter, the cells were stained with PE-conjugated anti-CD11b (Clone: M1/70, eBioscience) or PE-conjugated anti-CD68 (Clone: FA-11, AbD Serotec), and anti-Gpnmb (Clone: #297310, R&D Systems) followed by the addition of APC-conjugated secondary antibodies. Samples were evaluated using a CyAn ADP flow cytometer (Beckman Coulter) and analyzed with Kaluza software (Beckman Coulter).

Kumagai K., Tabu K., Sasaki F., Takami Y., Morinaga Y., Mawatari S., Hashimoto S., Tanoue S., Kanmura S., Tamai T., Moriuchi A., Uto H., Tsubouchi H, & Ido A. (2015). Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice. PLoS ONE, 10(11), e0143413.

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Characterizing Myeloid Cell Populations

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LMNCs were prepared as previously described. After Fc receptor blockade, the cells were stained with APC-conjugated anti-F4/80 (Clone: BM8, eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD11b (Clone: M1/70, eBioscience), and PE-conjugated anti-CD68 (Clone: FA-11, AbD Serotec, Raleigh, NC, USA). Alternatively, the cells were stained with FITC-conjugated anti-CD11b (Clone: M1/70, eBioscience), PE-conjugated anti-CD68 (Clone: FA-11, AbD Serotec), and anti-Gpnmb (Clone: #297310, R&D systems) followed by the addition of APC-conjugated secondary antibodies. Samples were evaluated using a CyAn ADP flow cytometer (Beckman Coulter, USA) and analyzed with Kaluza software (Beckman Coulter).

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Protein Extraction and Western Blot Analysis

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Proteins from the heart tissue of ISO treated, TAC mice, and control mice were harvested in buffer (50mM HEPES [pH 7.4], 150mM NaCl, 1% NP-40, 1mM EDTA, 1mM EGTA, 1mM glycerophosphate, 2.5mM sodium pyrophosphate 1mM Na3VO4, 20mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL of aprotinin, leupeptin, and pepstatin). Equal amounts of protein were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA) and transferred onto a nitrocellulose blot (Amersham, GE Healthcare). The blot was probed with the indicated primary antibodies using the polyclonal anti-GPNMB (R&D Systems, Minneapolis, MN) and anti-GAPDH (Invitrogen, Carlsbad, CA). Protein signals were detected using HRP conjugated secondary antibodies (Cell Signaling Technologies) and enhanced chemiluminescence (ECL) western blotting detection regents (Amersham, GE Healthcare).

Lin L.Y., Chun Chang S., O’Hearn J., Hui S.T., Seldin M., Gupta P., Bondar G., Deng M., Jauhiainen R., Kuusisto J., Laakso M., Sinsheimer J.S., Deb A., Rau C., Ren S., Wang Y., Lusis A.J., Wang J.J, & Huertas-Vazquez A. (2018). Systems Genetics Approach to Biomarker Discovery: GPNMB and Heart Failure in Mice and Humans. G3: Genes|Genomes|Genetics, 8(11), 3499-3506.

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