Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Flow cytometry [1]
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Validation data
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- Product number
- MA3-085 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- FXN Monoclonal Antibody (NPM-1B2)
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- MA3-085 detects Frataxin from human and mouse samples.
- Antibody clone number
- NPM-1B2
- Concentration
- Conc. Not Determined
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of Frataxin was performed by loading 20 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. Frataxin was detected at 23 kDa (human) and 19 kDa (mouse) using a Frataxin mouse monoclonal antibody (Product # MA3-085) at a dilution of 1:500 in blocking buffer for 1 hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of Frataxin, mitochondrial was achieved by transfecting HeLa with Frataxin, mitochondrial specific siRNAs (Silencer® select Product # S530862, S5362). Western Blot analysis (Fig. a) was performed using Membrane enriched extracts from the Frataxin, mitochondrial knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with FXN Monoclonal Antibody (NPM-1B2) (Product # MA3-085, 1:1000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). 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 Frataxin, mitochondrial.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Anti-FXN Monoclonal Antibody (NPM-1B2) (Product # MA3-085) and a 14 kDa band corresponding to Frataxin, mitochondrial was observed across cell lines tested. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1) and Jurkat (Lane 2) were electrophoresed using Novex™ 16% Tricine Protein Gel (Product # EC6695BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of Frataxin (green) in U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were stained with a Frataxin mouse monoclonal antibody (Product # MA3-085) at a dilution of 1:500 in staining buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG Superclonal™ Secondary Antibody, Alexa Fluor 488 conjugate (Product # A28175) at a dilution of 1:1000 for 1 hour at room temperature. Nuclei (blue) were counterstained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of Frataxin was done on U2OS cells. Cells were fixed, permeabilized and stained with a Frataxin mouse monoclonal antibody (Product # MA3-081, blue histogram) at a dilution of 1:100. After incubation of the primary antibody on ice for an hour, the cells were stained with a Goat anti-mouse IgG Secondary Antibody, DyLight 680 conjugate (Product # 35519) at a dilution of 1:50 for 1 hour on ice. A representative 10,000 cells were acquired for each sample. The black histogram represents unstained control cells.