Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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Validation data
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- Product number
- PA5-27874 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- GPNMB Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: SKBR3. Predicted reactivity: Rhesus Monkey (94%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- 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.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of GPNMB using 30 µg of PC-3 lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a GPNMB polyclonal antibody (Product # PA5-27874) at a dilution of 1:5000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot analysis of GPNMB was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a GPNMB Polyclonal Antibody (Product # PA5-27874) at a dilution of 1:500 and a HRP-conjugated anti-rabbit IgG secondary antibody.
- 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 Polyclonal Antibody (Product # PA5-27874, 1:2000) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000) and detected by chemiluminescence using the iBright™ FL1500 Imaging System (Product # A44115). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to GPNMB.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using GPNMB Polyclonal Antibody (Product # PA5-27874) and a 80 kDa band corresponding to GPNMB was observed across cell lines tested. Whole cell extracts (30 µg lysate) of SK-MEL-5 (Lane 1), U-87 MG (Lane 2), RT-4 (Lane 3), U-937 (Lane 4), SK-MEL-28 untreated (Lane 5), SK-MEL-28 treated with IFN gamma at 100 ng/mL concentration for 72 hrs (Lane 6), HT-29 (Lane 7) and HCT 116 (Lane 8) 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 (1:2000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).As shown here in the figure GPNMB levels of expression are reported to be low in HT-29 and HCT 116. As reported upon treatment with IFNγ, the levels of GPNMB expression should decrease, which was observed as shown in the figure.
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 Polyclonal Antibody (Product # PA5-27874, 1:100) in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32790, 1:2000), 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
- Immunohistochemical analysis of paraffin-embedded U87 xenograft, using GPNMB (Product # PA5-27874) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.