Overview

  • Product name
    Anti-NNT antibody [8B4BB10]
    See all NNT primary antibodies
  • Description
    Mouse monoclonal [8B4BB10] to NNT
  • Host species
    Mouse
  • Tested applications
    Suitable for: IP, ICC/IF, In-Cell ELISA, IHC-P, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Human heart mitochondria

  • 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.

    Product was previously marketed under the MitoSciences sub-brand.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

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.
In-Cell ELISA Use a concentration of 4 µg/ml. 0.4 µg/well
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

  • 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.
  • 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.
  • 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.
  • 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.
  • 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

This product has been referenced in:
  • Dey S  et al. Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes. J Biol Chem 291:11185-97 (2016). Read more (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 . Read more (PubMed: 23064950) »
See all 2 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Mar 27 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Dr. Amit Agarwal

Verified customer

Submitted Jul 18 2013

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

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

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

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.  

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Question
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.

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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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