- PA5-27874 - Invitrogen Antibodies GPNMB antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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)
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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)
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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.

PA5-27874