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Anti-NNT antibody [8B4BB10] (ab110352)

Reviews (2)Q&A (5)References (6)
Western blot - Anti-NNT antibody [8B4BB10] (ab110352)
  • Flow Cytometry - Anti-NNT antibody [8B4BB10] (ab110352)
  • Immunocytochemistry/ Immunofluorescence - Anti-NNT antibody [8B4BB10] (ab110352)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNT antibody [8B4BB10] (ab110352)
  • Immunoprecipitation - Anti-NNT antibody [8B4BB10] (ab110352)

Key features and details

  • Mouse monoclonal [8B4BB10] to NNT
  • Suitable for: IP, ICC/IF, IHC-P, WB, Flow Cyt
  • Reacts with: Mouse, Rat, Cow, Human
  • Isotype: IgG1

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 HRP

Overview

  • Product name

    Anti-NNT antibody [8B4BB10]
    See all NNT primary antibodies

  • Description

    Mouse monoclonal [8B4BB10] to NNT

  • Host species

    Mouse

  • Specificity

    Several customers have found that this antibody may not work well for mouse and rat samples.

  • Tested applications

    Suitable for: IP, ICC/IF, IHC-P, WB, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human

  • Immunogen

    Full length native protein (purified). This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • Normal Human skin fibroblasts (strain HDFn); Human cerebellum tissue; mitochondria from Human liver, Bovine heart, Rat liver and Mouse liver; HepG2 and HL-60 cells.

  • General notes

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

    Product was previously marketed under the MitoSciences sub-brand.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab110352 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
IP
Use at an assay dependent concentration.
ICC/IF
Use a concentration of 5 µg/ml.
IHC-P
1/100. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
WB (1)
Use a concentration of 0.5 µg/ml. Predicted molecular weight: 114 kDa.
Flow Cyt
Use a concentration of 1 µg/ml.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Notes
IP
Use at an assay dependent concentration.
ICC/IF
Use a concentration of 5 µg/ml.
IHC-P
1/100. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
WB
Use a concentration of 0.5 µg/ml. Predicted molecular weight: 114 kDa.
Flow Cyt
Use a concentration of 1 µg/ml.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Function

    The transhydrogenation between NADH and NADP is coupled to respiration and ATP hydrolysis and functions as a proton pump across the membrane.

  • Sequence similarities

    In the N-terminal section; belongs to the AlaDH/PNT family.
    In the C-terminal section; belongs to the PNT beta subunit family.

  • Cellular localization

    Mitochondrion inner membrane.
  • Information by UniProt

  • Database links

    • Alternative names

      • GCCD4 antibody
      • mitochondrial antibody
      • NAD(P) transhydrogenase antibody
      • NAD(P) transhydrogenase mitochondrial antibody
      • Nicotinamide nucleotide transhydrogenase antibody
      • NNT antibody
      • NNTM antibody
      • NNTM_HUMAN antibody
      • Pyridine nucleotide transhydrogenase antibody
      see all

    Images

    • Western blot - Anti-NNT antibody [8B4BB10] (ab110352)
      Western blot - Anti-NNT antibody [8B4BB10] (ab110352)
      Anti-NNT antibody [8B4BB10] (ab110352) at 0.5 µg/ml + HepG2 whole cell lysate

      Predicted band size: 114 kDa



      anti-Complex II 30kDa subunit has also been labeled as a control.
    • Flow Cytometry - Anti-NNT antibody [8B4BB10] (ab110352)
      Flow Cytometry - Anti-NNT antibody [8B4BB10] (ab110352)
      HL-60 cells were stained with 1 µg/mL NNT antibody (ab110352) (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.
    • Immunocytochemistry/ Immunofluorescence - Anti-NNT antibody [8B4BB10] (ab110352)
      Immunocytochemistry/ Immunofluorescence - Anti-NNT antibody [8B4BB10] (ab110352)
      Mitochondrial localization of NNT in cultured, normal Human skin fibroblasts (strain HDFn) labeled with anti NNT (ab110352) as the primary antibody and an AlexaFluor® 594 goat anti-mouse IgG1 secondary antibody.
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNT antibody [8B4BB10] (ab110352)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNT antibody [8B4BB10] (ab110352)
      NNT immunohistochemistry in Human cerebellum visualized with ab110352 at 1/100. NNT immunoactivity is most intense in neuronal cell bodies, most notably in the large Purkinje cells.
    • Immunoprecipitation - Anti-NNT antibody [8B4BB10] (ab110352)
      Immunoprecipitation - Anti-NNT antibody [8B4BB10] (ab110352)
      Nicotinamide nucleotide transhydrogenase (NNT) immunocaptured from Human liver mitochondria (lane 1), bovine heart mitochondria (lane 2), rat liver mitochondria (lane 3), mouse liver mitochondria (lane 4), and HepG2 cells (lane 5), using ab110352.

    References (6)

    Publishing research using ab110352? Please let us know so that we can cite the reference in this datasheet.

    ab110352 has been referenced in 6 publications.

    • Bharath LP  et al. Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation. Cell Metab 32:44-55.e6 (2020). PubMed: 32402267
    • Ward NP  et al. Nicotinamide nucleotide transhydrogenase regulates mitochondrial metabolism in NSCLC through maintenance of Fe-S protein function. J Exp Med 217:N/A (2020). PubMed: 32196080
    • McCambridge G  et al. Saturated Fatty Acid Activates T Cell Inflammation Through a Nicotinamide Nucleotide Transhydrogenase (NNT)-Dependent Mechanism. Biomolecules 9:N/A (2019). PubMed: 30823587
    • Ren W  et al. Decomposing the Mechanism of Qishen Granules in the Treatment of Heart Failure by a Quantitative Pathway Analysis Method. Molecules 23:N/A (2018). PubMed: 30041436
    • Dey S  et al. Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes. J Biol Chem 291:11185-97 (2016). PubMed: 27048652
    • Agarwal AR  et al. Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs. Am J Physiol Lung Cell Mol Physiol 303:L889-98 (2012). WB ; Mouse . PubMed: 23064950

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-7 of 7 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-NNT antibody [8B4BB10]

     Inconclusive
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    African green monkey Cell (COS-1)
    Permeabilization
    Yes - 0.2% TritonX-100
    Specification
    COS-1
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Mar 27 2017

    Western blot abreview for Anti-NNT antibody [8B4BB10 ]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    25 µg
    Gel Running Conditions
    Reduced Denaturing (10)
    Sample
    Mouse Tissue lysate - other (Lung mitochondrion)
    Specification
    Lung mitochondrion
    Blocking step
    Casein-TBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Dr. Amit Agarwal

    Verified customer

    Submitted Jul 18 2013

    Question

    Hi,

    Can you please let me know when you estimate that you will get some feedback on this enquiry.

    I would like to get back to the customer with some indication of how long it will be, thanks

    Kind regards,

    Read More
    Answer

    Thank you for following up on this query.

    The staining displayed on the datasheet of the Anti-NNT antibody [8B4BB10] is not from an Abreview but from testing we have commissioned ourselves.

    This was performed on human cerebellum tissue, with antigen retrieval being performed using 1 mmol EDTA, pH8 in pressure cooker. The signal of the primary antibody was developed using a polymer-detection system and visualised with DAB.

    The staining of the NNT in the tissue is therefore marked by brown colour whilst the nucleus of the cells have been counter stained blue. If there were cells with no target in them thenthey would be expected to not have any colour apart from the blue counter stain.

    The customer may need to optimise the antigen retrieval and staining protocol in order to achieve optimal staining. If the customer would like help with this could they please fill out the questionnaire I have attached to this email.

    I hope this information has been of help.If you have any further questions, please do not hesitate to ask.

    Read More

    Question

    Customer kindly called to ask whether we had any data regarding the use of ab110352, anti-NNT antibody [8B4BB10] in mouse whole cell lysates as the information on the datasheet shows only mitochondrial isoloates.

    Read More
    Answer

    Thank you for contacting us.

    I have had the opportunity to speak with the lab regarding to the antibody ab110352 [8B4BB10], we only tested human whole cell lysate (HepG2) not mouse cell lysate. This antibody has low affinity to mouse NNT based on the IP result. You may try with mouse whole cell lysates, but the signal may be very low comparing to human or rat cell lysates.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

    Read More

    Question

    The positive control lysate did not show a signal in my western blot. Is it possible for a new antibody to be sent to me?

    Read More
    Answer

    I am sorry that you still did not receive a signal with the original antibody when using the positive control that I sent. I have processed you request to receive a new vial of ab110352 and you should have it by Friday of this week. I am sorry for the delay; however we will try and get it to you as soon as possible. The problem is that we only have two vials and they are in our UK office and so we have to have the antibody transferred to the US. Your new order number is *******. Please let me know if you have any issues with the new vial of antibody and I do recommend using the positive lysate the first time you try the new

    Read More

    Question

    I talked to my Professor and we will try another vial of the NNT that you can provide.     I am going to see if another lab has the recommended positive controls.     Thank  you for your help.  I look forward to trying the new antibody.  

    Read More
    Answer

    Thank you for confirming these details and for your cooperation. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 978308. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

    Read More

    Question

    LOT NUMBER GR47028-1 ORDER NUMBER xxxxxx DESCRIPTION OF THE PROBLEM Non-specific band SAMPLE mouse liver mitochondria PRIMARY ANTIBODY Primary in same blocking buffer concentration at 1:1000 dilution as recommended by Abcam/mitoscience and then again we increased both the protein and concentration to 1:250 dilution. this was incubated over night in the fridge. Washed with TTBS buffer 3X for 15 minutes each DETECTION METHOD Immunstar HRP substrate for 5minutes and immediate exposure POSITIVE AND NEGATIVE CONTROLS USED none used ANTIBODY STORAGE CONDITIONS at 4 degrees SAMPLE PREPARATION tried 20 and 50µg of protein, with sample buffer with 2betamercaptoethanol with 5 minutes of boiling of sample with buffer. AMOUNT OF PROTEIN LOADED tried 20µg didn't work so we increased to 50µg of protein. both gave non specific bands lower than the correct kDa ELECTROPHORESIS/GEL CONDITIONS used tris/glycine gels from Biorad at 10-12% gels TRANSFER AND BLOCKING CONDITIONS transfer to PVDF membrane and blocked with 5% milk in TBS buffer with 0.05% tween. one hour at room temperature SECONDARY ANTIBODY Used Goat anti mouse HRP conjugate in blocking buffer concentration and was done for one hour at room temperature. Washed 3X in TTBS buffer 3X for 15 minutes each HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? we increased protein and concentration of the primary antibody.

    Read More
    Answer

    Thank you for contacting us and reporting this problem. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. The information provided by you will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. Having reviewed the protocol I have a few things I'd like to clarify: 1. was the membrane aggitated in the fridge with the incubation of the primary antibody? 2. could you describe how the mouse liver mitochondria sample is being prepared? 3. were protease inhibitors used in the lysis buffer? 4. what non-specific band are being seen? Could you share an image of the blot if possible? 5. has the transfer of the proteins been confirmed by staining of the membrane? 6. What dilution was used for the secondary antibody I look forward to recieving your reply.

    Read More

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