Overview

  • Product name
  • Description
    Rabbit polyclonal to mtTFA
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human mtTFA.

    Read Abcam's proprietary immunogen policy (Peptide available as ab47516.)

  • Positive control
    • This antibody gave a positive signal in the following whole cell lysates: HeLa, Jurkat, A431. This antibody gave a positive signal in the following tissue lysates: Skeletal Muscle (Human) - adult normal tissue, Heart (Human) - adult normal tissue, Brown adipose (Mouse), Brown adipose (Rat), Heart (Mouse), Heart (Rat).

Properties

Applications

Our Abpromise guarantee covers the use of ab47517 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P 1/750. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 25 kDa (predicted molecular weight: 29 kDa).
ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    Binds to the mitochondrial light strand promoter and functions in mitochondrial transcription regulation. Required for accurate and efficient promoter recognition by the mitochondrial RNA polymerase. Promotes transcription initiation from the HSP1 and the light strand promoter by binding immediately upstream of transcriptional start sites. Is able to unwind and bend DNA. Required for maintenance of normal levels of mitochondrial DNA. May play a role in organizing and compacting mitochondrial DNA. target DNA. Interacts with TFB1M and TFB2M.
  • Sequence similarities
    Contains 2 HMG box DNA-binding domains.
  • Cellular localization
    Mitochondrion.
  • Information by UniProt
  • Database links
  • Alternative names
    • anscription factor 6-like 1 antibody
    • Mitochondrial transcription factor 1 antibody
    • mitochondrial transcription factor A antibody
    • MtTF1 antibody
    • mtTFA antibody
    • TCF 6 antibody
    • TCF-6 antibody
    • TCF6 antibody
    • TCF6L1 antibody
    • TCF6L2 antibody
    • TCF6L3 antibody
    • TFAM antibody
    • TFAM_HUMAN antibody
    • Transcription factor 6 antibody
    • Transcription factor 6 like 2 (mitochondrial transcription factor) antibody
    • Transcription factor 6 like 2 antibody
    • Transcription factor 6-like 2 antibody
    • transcription factor 6-like 3 antibody
    • Transcription factor A, mitochondrial antibody
    • Transcription factor A, mitochondrial antibody
    • Transcription factor A, mitochondrial precursor antibody
    see all

Images

  • All lanes : Anti-mtTFA antibody (ab47517) at 1 µg/ml

    Lane 1 : Skeletal Muscle (Human) Tissue Lysate - adult normal tissue
    Lane 2 : Human heart tissue lysate - total protein (ab29431)
    Lane 3 : Brown adipose (Mouse) Tissue Lysate
    Lane 4 : Brown adipose (Rat) Tissue Lysate
    Lane 5 : Heart (Mouse) Tissue Lysate
    Lane 6 : Heart (Rat) Tissue Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 29 kDa
    Observed band size: 29 kDa
    Additional bands at: 100 kDa, 18 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 8 minutes


    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab47517 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • ICC/IF image of ab47517 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab47517, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

  • IHC-P image of mtTFA staining using ab47517 in kidney (upper) and Colon (lower) sections. The tissue was fixed using formaldehyde. Heat mediated epitope retrieval was carried out using citric acid (pH 6). The sections were blocked uwing 1% BSA for 10 mins at 21°C.

    See Abreview

  • All lanes : Anti-mtTFA antibody (ab47517) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 29 kDa
    Observed band size: 25 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.

  • All lanes : Anti-mtTFA antibody (ab47517) at 2 µg/ml

    Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Human mtTFA peptide (ab47516) at 2 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 29 kDa
    Observed band size: 24,26 kDa why is the actual band size different from the predicted?
    Additional bands at: 50 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 10 minutes

References

This product has been referenced in:
  • Masuike Y  et al. Esophageal squamous cell carcinoma with low mitochondrial copy number has mesenchymal and stem-like characteristics, and contributes to poor prognosis. PLoS One 13:e0193159 (2018). Read more (PubMed: 29447301) »
  • Torralba D  et al. Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts. Nat Commun 9:2658 (2018). Read more (PubMed: 29985392) »
See all 12 Publications for this product

Customer reviews and Q&As

1-4 of 4 Q&A

Answer

Thank you for confirming the results of the customer. Unfortunately, Anja is not in the office at present and is therefore unable to help resolve this case but I will try my best in her place.

I think the results look much improved with the blocking peptide. Think continuing using the 5% BSA blocking and perhaps reducing the exposure time used should lead to further improvements. I can understand that your customer may be frustrated that he has been using up a lot of antibody to perform these optimisations and if he would like I can offer to send a free of charge vial of ab47517. If you would like for me to organise this please do let me know.

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Question

LOT NUMBER GR30593-1 ORDER NUMBER xx DESCRIPTION OF THE PROBLEM Multiple bands (can’t distinguish which one was right) SAMPLE - Species: human - What’s cell line or tissue: primary fibroblast cell (from the newborns) - Cell extract or Nuclear extract: cell extract - Purified protein or Recombinant protein: Whole cell extraction PRIMARY ANTIBODY - Species: rabbit polyclonal - Reacts against: human - At what dilution(s) have you tested this antibody: 1:900 - What dilution buffer was used: 5% non-fat-milk in TBST - Incubation time: 1hr - Incubation temperature: room temperature - What washing steps were done: 15min x 1 + 5min x 3 DETECTION METHOD ECl+ ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION - What lysis buffer was used: 20mM potassium phosphate buffer + sonication - What protease inhibitors were used: cocktail protease inhibitor - What loading buffer was used: NuPAGE LDS sample buffer (invitrogen:Cat no.NP0007) - Phosphatase inhibitors - Did you heat the samples: temperature and time: 70℃ for 10min AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS - Reducing or non reducing gel: Reducing - Reducing agent: 2-ME - Gel percentage : 4-12% TRANSFER AND BLOCKING CONDITIONS - Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, Coomassie blue (Invitrogen iBlot dry blotting system Cat no. IB10001) Blocking conditions - Buffer: 5% non-fat-milk in TBST - Blocking agent: milk, BSA, serum, what percentage: 5% - Incubation time: 1hr - Incubation temperature: room temperature SECONDARY ANTIBODY - Species: goat - Reacts against: rabbit - At what dilution(s) have you tested this antibody: 1:5000 - Incubation time: 1hr - Wash steps: 15min x 1 + 5min x 3 - Fluorochrome or enzyme conjugate: enzyme conjugate - Do you know whether the problems you are experiencing come from the secondary? HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

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Answer

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with a vial of anti-mtTFA antibody ab47517.

We have received no complaints about this product therefore I am concerned it is not working for you. I appreciate the time taken to submit further information to us and I would like to make some suggestions to the protocol in an attempt to improve the results obtained:

When testing this antibody, our lab used 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands between 25 and 34 kDa. An example of the above (BSA instead of milk)is the western blot obtained with the Abcam GAPDH antibody ab9385 (https://www.abcam.com/index.html?datasheet=9385 (or use the following: https://www.abcam.com/index.html?datasheet=9385).)

I hope this suggestion will improve the results. As the antibody was purchased inDecember you are covered by our Abpromise guarantee. Therefore if the suggestion does not improve the results I would be happy to offer a refund or replacement vial. Please do not hesitate to contact me should you have additional questions.

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Answer

Thank you for contacting us. My colleague "Ariana" has already sent her reply so I am closing this case. The antibodies are fine for 1 week at room temperature however in your case if the products fail to perform I would suggest getting refund from insurance company. You can get information of product price from our website at  https://www.abcam.com/ I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.    

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Answer

Thank you for contacting us. In order to help you with your question could you provide the following - Order number of purchase - for how long the power cut happened? -Were these products at room temperature when the electricity switched on again? What do you really want us to do? Do you need quote for these products please explain? I look forward to hearing from you soon.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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