- PA5-27865 - Invitrogen Antibodies TFAM antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using TFAM Polyclonal Antibody (Product # PA5-27865). Sample (30 µg of whole cell lysate). Lane A: 293T. Lane B: A431. Lane C: HeLa. Lane D: HepG2. 12% SDS PAGE. TFAM Polyclonal Antibody (Product # PA5-27865) diluted at 1:5,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of mtTFA was achieved by transfecting HeLa cells with mtTFA specific siRNAs (Silencer® select Product # s14000, s14001). Western blot analysis (Fig. a) was performed membrane enriched extracts from the HeLa knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with mtTFA Polyclonal Antibody (Product # PA5-27865, 1:5000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 mtTFA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-mtTFA Rabbit Polyclonal Antibody (Product # PA5-27865) and 26 kDa, 29 kDa bands corresponding to mtTFA were observed in cell lines and tissues tested along with an uncharacterized band (*) in Rat Heart. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), T-47D (Lane 2), MDA-MB-231 Lung (Lane 3), Caco-2 (Lane 4), Mouse Heart (Lane 5), Rat Heart (Lane 6) and Mouse Lung (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 (1:5000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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: Immunocytochemistry-Immunofluorescence analysis of TFAM was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: TFAM Polyclonal Antibody (Product # PA5-27865) diluted at 1:1000. Red: alpha Tubulin, a cytoskeleton marker. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: TFAM Polyclonal Antibody detects mtTFA protein at mito by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: mtTFA stained by TFAM Polyclonal Antibody (Product # PA5-27865) diluted at 1:500. Red: alpha Tubulin, a cytoskeleton marker, stained by alpha Tubulin Polyclonal Antibody [GT114] (Product # MA5-31466) diluted at 1:1,000. Blue: Fluoroshield with DAPI .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of mtTFA was performed using Caco-2 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 mtTFA Rabbit Polyclonal Antibody (Product # PA5-27865) at 1:500 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 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). Panel d represents the composite image showing mitochondrial pattern of mtTFA. 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: Immunohistochemistry (Paraffin) analysis of TFAM was performed in paraffin-embedded human esophagus cancer tissue using TFAM Polyclonal Antibody (Product # PA5-27865) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry (Paraffin) analysis of TFAM was performed in paraffin-embedded human colon cancer tissue using TFAM Polyclonal Antibody (Product # PA5-27865) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: TFAM Polyclonal Antibody immunoprecipitates mtTFA protein in IP experiments. IP Sample: 293T whole cell lysate/extract A. 40 µg 293T whole cell lysate/extract B. Control with 2 µg of preimmune rabbit IgG C. Immunoprecipitation of mtTFA protein by 2 µg of TFAM Polyclonal Antibody (Product # PA5-27865) 15% SDS-PAGE The immunoprecipitated mtTFA protein was detected by TFAM Polyclonal Antibody (Product # PA5-27865) diluted at 1:1,000.

PA5-27865