MA5-49360

antibody from Invitrogen Antibodies

Targeting: GPNMB HGFIN, NMB

Provider product page for MA5-49360

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Antibody data

Antibody Data
Antigen structure
References [0]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [4]
Immunohistochemistry [1]
Flow cytometry [1]

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Product number: MA5-49360 - Provider product page
Provider: Invitrogen Antibodies
Product name: GPNMB Recombinant Rabbit Monoclonal Antibody (PSH0-82)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: Sequence Similarities: 98% Mouse/Rat. Tissue Specificity: Widely expressed, but very low expression, if any, in the brain (PubMed:12609765, PubMed:16609006). Expressed in the epidermis with higher levels in melanocytes compared with keratinocytes and Langerhans cells (at protein level) (PubMed:29336782). Expressed in peripheral blood, but not bone marrow mononuclear cells (PubMed:12609765). Expressed in tissue macrophages, including liver Kuppfer cells and lung alveolar macrophages, in podocytes and in some cells of the ciliary body of the eye (at protein level) (PubMed:16489096). May be overexpressed in various cancers, including melanoma and glioblastoma multiforme (PubMed:7814155, PubMed:16489096, PubMed:16609006). Positive Control: L929 cell lysate, A375 cell lysate, U-937 cell lysate, rat heart tissue lysate, B16F1 cell lysates, human lung tissue, L929. Subcellular Location: Cell membrane, Melanosome membrane, Early endosome membrane. Predicted band size: 64 kDa.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Antibody clone number: PSH0-82
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

GPNMB protein structure - MA5-49360 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of GPNMB was achieved by transfecting SK-MEL-5 with GPNMB specific siRNAs (Silencer® select Product # s20461, s20462). Western blot analysis (Fig. a) was performed using whole cell extracts from the GPNMB knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360, 1:2,000 dilution ) and Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to GPNMB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) and, 110 kDa and 80 kDa bands corresponding to GPNMB were observed across high expressing cell lines and not in low expressing cell lines tested. Whole cell extracts (30 µg lysate) of SK-MEL-5 (Lane 1), RT4 (Lane 2), U-937 (Lane 3), SK-MEL-28 untreated (Lane 4), SK-MEL-28 treated with IFN gamma at 100 ng/mL concentration for 72 hours (Lane 5), low expressing cell lines such as HT-29 (Lane 6) and HCT 116 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2,000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34579). In lane 5, upon treatment with IFN gamma, GPNMB expression decreases in malignant melanoma as reported.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) and 80-120 kDa bands corresponding to GPNMB was observed across cell lines tested. Whole cell extracts (20 µg lysate) of L929 (Lane 1), A375 (Lane 2), U-937 (Lane 3) and Whole cell extracts (40 µg lysate) of Rat heart (Lane 4) were electrophoresed using 4-20% SDS-PAGE gel. Resolved proteins were transferred onto a PVDF membrane. The blot was blocked with 5% NFDM/TBST for 1 hour at room temperature, then probed with the primary antibody (1:1,000 dilution) for 2 hours at room temperature and detected by chemiluminescence with HRP labeled Goat anti-Rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) and 80-120 kDa bands corresponding to GPNMB was observed across cell lines tested. Whole cell extract (20 µg lysate) of B16F1 was electrophoresed using 4-20% SDS-PAGE gel. Resolved proteins were transferred onto a PVDF membrane. The blot was blocked with 5% NFDM/TBST for 1 hour at room temperature, then probed with the primary antibody (1:1,000 dilution) for 2 hours at room temperature and detected by chemiluminescence with HRP labeled Goat anti-Rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GPNMB was performed using 70% confluent log phase SK-MEL-28 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32790), (1:2,000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cell membrane and cytosolic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GPNMB using L929 cells. The cells were fixed 4% paraformaldehyde for 10 minutes, permeabilized with 0.05% Triton™ X-100 in PBS for 20 minutes, and blocked with 2% negative goat serum for 30 minutes at room temperature. The cells were labeled with GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) at 1:100 dilution in 2% negative goat serum overnight at 4°C and then with iFluor™ 488 Goat Anti-Rabbit IgG H&L secondary antibody (1:1,000 dilution) for 1 hour at room temperature (green). Nuclear were stained with DAPI (blue). Beta tubulin (red) was stained at 1:200 dilution overnight at +4°C. iFluor™ 594 Goat Anti-Mouse IgG H&L was used as the secondary antibody at 1:1,000 dilution. The images were captured at 200X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GPNMB was performed using 70% confluent log phase SK-MEL-5 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32790), (1:2,000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cell membrane and cytosolic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GPNMB was performed using 70% confluent log phase B16-F10 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with GPNMB Rabbit Recombinant Monoclonal Antibody (PSH0-82) (Product # MA5-49360) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32790), (1:2,000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cell membrane and cytosolic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.