- 45-6300 - Invitrogen Antibodies FXN antibody

Punga T, Bühler M
EMBO molecular medicine 2010 Apr;2(4):120-9

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of FXN in recombinant protein using a FXN Monoclonal antibody (Product # 45-6300) at a concentration of 5 µg/mL. 0.0005 µg of recombinant Human Frataxin was run in this analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of FXN in recombinant protein using a FXN Monoclonal antibody (Product # 45-6300) at a concentration of 5 µg/mL. 0.0005 µg of recombinant Human Frataxin was run in this analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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 (18A5DB1) (Product # 45-6300, 3 µg/mL ) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-FXN Monoclonal Antibody (18A5DB1) (Product # 45-6300) and a 14 kDa band corresponding to Frataxin, mitochondrial was observed across cell lines tested. Membrane enriched extracts (30 µg lysate) of Jurkat (Lane 1), A-431 (Lane 2) and HeLa (Lane 3) 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 (3 µg/mL) 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemical analysis of FXN in MRC5 using a FXN Monoclonal antibody (Product # 45-6300) at a concentration of 0.5 µg/mL. The normal Human fibroblasts (MRC5) were fixed and permeabilized, and detected using an AlexaFluor® 594-conjugated-goat-anti-mouse IgG1 isotype specific secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of FXN in Human colon adenocarcinoma tissue sections using a FXN Monoclonal Antibody (Product # 45-6300).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of FXN in HL60 cells using an FXN monoclonal antibody (Product # 45-6300) at 1 µg/mL is depicted by the blue line. The red line indicates an isotype control antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Pathogenic GAA repeat expansions reduce FXN mRNA and protein levels Schematic view of the human FXN gene (ENSG00000165060). Exons and untranslated regions are depicted as black and white boxes, respectively. The red arrowhead indicates localization of the GAA repeats. Northern blot and TaqMan probes annealing to the FXN mRNA are indicated as blue and red lines, respectively. Cell lines used throughout this study. All cell lines were obtained from Coriell Cell Repositories. Reduced accumulation of FXN mRNA in FRDA cells as determined by qRT-PCR. Values for FXN mRNA were normalized to 18S rRNA. Northern blot showing that no major alternatively spliced FXN isoforms exist and that mRNA levels are reduced in FRDA cells. The Northern blot was probed with a human FXN cDNA probe. 18S served as a loading control. Western blot showing reduced accumulation of FXN protein in FRDA cells. Intermediate (i) and mature (m) forms of the FXN proteins are indicated.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 H3K9 methylation is locally confined in FRDA cells and does not affect marks of transcription initiation ChIP assay to profile H3K9me3 along the FXN gene. The profile for wild type is shown in blue (

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Expanded GAA repeats affect H3K36 trimethylation Treatment of wild-type cells with the transcription elongation inhibitor DRB reduces the H3K36me3 signal along the FXN gene locus. Wild-type (GM15851) cells were treated with 115 uM DRB for 5 h before ChIP with anti-H3K36me3 antibody. Location of qPCR probes and fold enrichment is presented as in Fig 3A . Reduced accumulation of FXN mRNA after DRB treatment in wild-type (GM15851) cells. RNA was isolated from the same cells as used for the ChIP shown in (A). ChIP assay to profile H3K36me3 along the FXN gene in wild type and different FRDA cell lines. The profile for wild type is shown in blue (

45-6300