Overview

  • Product name

  • Description

    Rabbit polyclonal to ND6
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WBmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Pig, Chimpanzee
  • Immunogen

    Synthetic peptide corresponding to Human ND6 aa 110-159 (internal sequence).
    Sequence:

    DGVVVVVNFNSVGSWMIYEGEGSGLIREDPIGAGALYDYGRWLVVVTGWT


    Database link: NP_536854

  • Positive control

    • Human fetal lung lysate.
  • General notes

     This product was previously labelled as NADH Dehydrogenase subunit 6

     

Properties

Applications

Our Abpromise guarantee covers the use of ab81212 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 Use a concentration of 1 µg/ml.
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 19 kDa. Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

Target

  • Relevance

    NADH Dehydrogenase subunit 6 (MTND6) is 1 of the 7 mitochondrial DNA (mtDNA) encoded subunits (MTND1, MTND2, MTND3, MTND4L, MTND4, MTND5, MTND6) included among the approximately 41 polypeptides of respiratory Complex I. Complex I accepts electrons from NADH, transfers them to ubiquinone (Coenzyme Q10), and uses the energy released to pump protons across the mitochondria inner membrane. MTND6 has been proposed to be a component of the iron-protein fragment. The predicted polypeptide has a molecular weight of 18.6 kD. Its apparent MW on SDS-polyacrylamide gels (PAGE) using Tris-glycine buffer is 16.7 kD, and the apparent MW on urea-phosphate gels is also close to the predicted molecular weight.
  • Cellular localization

    Mitochondrion membrane; Multi-pass membrane protein.
  • Database links

  • Alternative names

    • Mitochondrially encoded NADH dehydrogenase 6 antibody
    • MT ND6 antibody
    • mtND6 antibody
    • NADH dehydrogenase subunit 6 (complex I) antibody
    • NADH ubiquinone oxidoreductase chain 6 antibody
    • NADH Ubiquinone Oxidoreductase subunit ND6 antibody
    • NADH6 antibody
    • ND6 antibody
    see all

Images

  • Anti-ND6 antibody (ab81212) at 1 µg/ml (in 5% skim milk / PBS buffer) + Human fetal lung lysate at 10 µg

    Secondary
    HRP conjugated anti-Rabbit IgG at 1/50000 dilution

    Predicted band size: 19 kDa
    Observed band size: 24 kDa
    why is the actual band size different from the predicted?



    Gel concentration: 12%
  • IHC image of ab81212 staining in Human breast cancer formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab81212, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:

  • Zhang Q  et al. Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function. Nucleic Acids Res 46:10930-10945 (2018). Read more (PubMed: 30137487) »
  • Zhu G  et al. Acute effect of lactic acid on tumor-endothelial cell metabolic coupling in the tumor microenvironment. Oncol Lett 12:3478-3484 (2016). WB ; Human . Read more (PubMed: 27900024) »
See all 6 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (macrophage)
Specification
macrophage
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 22 2014

Application
Western blot
Loading amount
100 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Cell lysate - whole cell (MDM)
Specification
MDM
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Nov 27 2013

Question

The samples are prepared from schwann cells and DRG neurons and pancreatic cancer cell lines (one human, one rat origin, one mouse origin ) we chose the antibodies according to the reactivity mentioned on the abcam data sheet and the lysates were prepared using a non denaturing lysis buffer, aliquoted and stored at -80 degrees centigrade. I use 10% and 12.5% acrylamide gels depending on the molecular weight of the protein. initially 40 ug protein was loaded and the gels were run at 70 volts and transfered on to PVDF membrane (ice cold). All buffers were prepared as suggested in the western blot guide by Abcam. 5% milk was used to block the membrane, washed thrice with TBST and/ or TBS. Incubated with respective antibodies (initially 1: 1000 dilution) overnight in cold room with agitation. The blots were added with secondary antibody the following day (secondary antibody from abcam (ab97023 and ab97051 which were aliquoted and stored at -20 degrees centigrade) incubated for 1 hour at room temperature and then washed thrice with TBS/and or TBST. probed with chemiluminescent solution from Biorad. Bands were not detected. We tried loading 50ug protein (we cannot load more owing to the limited volumes of sample we have and the nature of the project) and then tried using 1:500 and 1:250 dilutions, but still the antibodies were not working. We have been using this protocol and works fine with all the other abcam antibodies. Please let me know if I need to elaborate on any of these steps.

Read More
Answer


Upon reviewing your protocol, it is troubling to me that all 4 of these antibodies are not working. While it is conceivable that all four antibodies are not performing properly, typically when multiple antibodies are not working it is an issue with sample preparation, membrane transfer, or the detection substrate. In your case, I think the following troubleshooting steps are the most worth your while attempting, with tip 1 being the first thing you try since it is the most likely to make the targets recognizable by the antibodies:
1. You mentioned your samples were non-denatured. ab2571, ab32124, and ab81212 were all raised against synthetic peptides and will require linear proteins in order to recognize their epitopes. Thus, your samples should be both reduced and denatured (SDS and beta-mercaptoethanol or DTT) and boiled for 5 minutes at 100C prior to freezing at -20C. RIPA buffer is a good choice for mitochondrial and membrane proteins such as these.
2. To enrich the target proteins, have you considered a mitochondria isolation kit such as ab110170 (https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html)?
3. Have you insured that the proteins are being transferred with a reversible stain such as Ponceau S? Since you are using PVDF, make sure to pre-soak the membrane in MeOH, then in transfer buffer.
4. If you are including milk in your antibody dilutions try removing milk or reducing to 0.5% milk for the antibody dilutions only.
Further troubleshooting tips, including no signal specific tips, are included through the link below:
https://www.abcam.com/index.html?pageconfig=resource&rid=11352
Please let me know the results of troubleshooting, especially step 1. I look forward to your reply and sincerely hope this information helps.

Read More
Application
Western blot
Sample
Fruit fly (Drosophila melanogaster) Tissue lysate - whole (Whole larval extract)
Loading amount
100 µg
Specification
Whole larval extract
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Nov 05 2012

Application
Western blot
Sample
Human Cell lysate - other (Mitochondrial fractions of HeLa WT and HeLa rho-0)
Loading amount
26 µg
Specification
Mitochondrial fractions of HeLa WT and HeLa rho-0
Gel Running Conditions
Reduced Denaturing (Reduced Denaturing (Reduced Denaturing (4-15%))
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 21 2012

Question

Dear Technical
Order Details:




Antibody code: ab81212

Batch number: GR62732-1

General Information:

Antibody storage conditions (temperature/reconstitution etc)

Stored at -20C.

Description of the problem (high background, low signal, non-specific staining etc.)

No staining and background.

Sample (Species/Tissue/Cell Type/Cell Line etc.)

1. Human Breast cancer.

2. Mouse liver implanted with human cells.

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)

Paraformaldehyde for about 2 days at room temp.

Prepared paraffin embedded slides from the sample.

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Heat mediated Citrate buffer.

Permeabilization step

Blocking conditions (Buffer/time period, Blocking agent etc.)

Primary Antibody (Diluent/Dilution/Incubation time, Wash step)

Diluted at 1:1000 and 1:300 in Invitrogen green antibody diluent, Cat # 00-3118.

Incubated for 1 hr at room temp.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

Super Picture Ploy HRP conjugate from Invitrogen, Cat # 87-8963.

Detection method

DAB staining

Positive and negative controls used (please specify)

Positive control – Human breast cancer

Optimization attempts (problem solving):

How many times have you tried the IHC?

1

Have you run a "No Primary" control?

No

Do you obtain the same results every time?

Yes

What steps have you altered?

Additional Notes

Document Attachment: images of the results may be very helpful. If you wish to add it to the complaint form, please attach them to the reply e-mail..

Read More
Answer

Thank you for your enquiry regarding ab81212 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

The protocol looks fine to me, however it would be much appreciated if I could get some more information which would help me identify the source of the problem.

1) Antigen retrieval: I understand that one experiment has been carried out only and the result is negative. Could you please let me know how long and at what pH the demasking was performed? Has any optimization been tried?

2) Incubation: Has the customer tried longer incubation such as 2 hrs or overnight to see if the signal is getting stronger at all?

3) Permeabilization: mtND6 (NADH Dehydrogenase subunit 6) is localized in the Mitochondrion membrane so permeabilization of the membrane may be important. I am not sure what the green antibody diluent contains?

4) Detection system: Is the secondary antibody compatible with the primary antibody (rabbit IgG)? Could you please confirm? Does the detection system work fine? Have you used it successfully with another primary antibody? Is the HRP still active?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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