- PA5-29571 - Invitrogen Antibodies TFAM antibody

Chen YJ, Chang CF, Angayarkanni J, Lin WT
Molecules (Basel, Switzerland) 2021 Oct 30;26(21)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of mtTFA using Various whole cell extracts (30 µg). Samples were loaded onto a 12% SDS-PAGE gel and probed with a mtTFA polyclonal antibody (Product # PA5-29571) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of TFAM was performed by separating 30 µg of whole cell extract by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a TFAM Polyclonal Antibody (Product # PA5-29571) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using TFAM Polyclonal Antibody (Product # PA5-29571). Various whole cell extracts (30 µg) were separated by 12% SDS-PAGE, and the membrane was blotted with TFAM Polyclonal Antibody (Product # PA5-29571) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using TFAM Polyclonal Antibody (Product # PA5-29571). Various whole cell extracts (30 µg) were separated by 12% SDS-PAGE, and the membrane was blotted with TFAM Polyclonal Antibody (Product # PA5-29571) diluted at 1:500. 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 # PA5-29571, 1:1000 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 # PA5-29571) and 26 kDa, 29 kDa bands corresponding to mtTFA were observed in cell lines and tissues tested except Mouse Lung and Mouse Spleen which are reported to be negative. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), T-47D (Lane 2), MDA-MB-231 (Lane 3), Caco-2 (Lane 4), Mouse Heart (Lane 5), Rat Heart (Lane 6), Mouse Lung (Lane 7) and Mouse Spleen (Lane 8) 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 (1:1000 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 # PA5-29571) diluted at 1:500. Red: Mitotracker, a mitochondria marker. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of TFAM was performed in NT2D1 cells fixed in 2% paraformaldehyde/culture medium at RT for 30 min. Green: TFAM Polyclonal Antibody (Product # PA5-29571) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: TFAM antibody detects TFAM protein at mitochondria by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: TFAM stained by TFAM antibody (Product # PA5-29571) diluted at 1:1,000.

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 # PA5-29571) at 1:100 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: Immunohistochemical analysis of paraffin-embedded human breast cancer, using mtTFA (Product # PA5-29571) antibody at 1:250 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

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 # PA5-29571) 12% SDS-PAGE The immunoprecipitated mtTFA protein was detected by TFAM Polyclonal Antibody (Product # PA5-29571) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 Effect of PPH902 on the activation of mitochondrial biogenesis: Representative Western blotting shows changes in the levels of NRF1 and TFAM in C2C12 cells. *** p < 0.001 indicates a significant difference with respect to control groups and ## p < 0.01 and ### p < 0.001 indicates a significance difference with respect to the high glucose challenge groups.

PA5-29571