Overview

  • Product name
  • Description
    Mouse polyclonal to Ndufs1
  • Host species
    Mouse
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Mouse
    Predicted to work with: Rat, Chicken, Cow, Human, Xenopus laevis, Chimpanzee, Zebrafish, Cynomolgus monkey, Gorilla, Chlamydomonas reinhardtii, Orangutan
  • Immunogen

    Synthetic peptide:

    MFGSDRSRFLEGKRAVEDKN

    , corresponding to amino acids 145-165 of Ndufs1

  • General notes


    This antibody was raised by a genetic immunization technique. Genetic immunization can be used to generate antibodies by directly delivering antigen-coding DNA into the animal, rather than injecting a protein or peptide (Tang et al. PubMed: 1545867; Chambers and Johnston PubMed 12910245; Barry and Johnston PubMed: 9234514). The animal's cells produce the protein, which stimulates the animal's immune system to produce antibodies against that particular protein. A vector coding for a partial fusion protein was used for genetic immunisation of a mouse and the resulting serum was tested in Western blot against an E.coli lysate containing that partial fusion protein. Genetic immunization offers enormous advantages over the traditional protein-based immunization method. DNA is faster, cheaper and easier to produce and can be produced by standard techniques readily amenable to automation. Furthermore, the antibodies generated by genetic immunization are usually of superior quality with regard to specificity, affinity and recognizing the native protein.

Properties

Applications

Our Abpromise guarantee covers the use of ab52690 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
WB 1/1000. Detects a band of approximately 32.2 kDa (predicted molecular weight: 79 kDa).

This antibody has been tested in Western blot against an E.coli lysate containing the partial recombinant fusion protein used as an immunogen. We have no data on detection of endogenous protein.

Target

  • Function
    Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that is believed to belong to the minimal assembly required for catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone (By similarity). This is the largest subunit of complex I and it is a component of the iron-sulfur (IP) fragment of the enzyme. It may form part of the active site crevice where NADH is oxidized.
  • Involvement in disease
    Defects in NDUFS1 are a cause of mitochondrial complex I deficiency (MT-C1D) [MIM:252010]. A disorder of the mitochondrial respiratory chain that causes a wide range of clinical disorders, from lethal neonatal disease to adult-onset neurodegenerative disorders. Phenotypes include macrocephaly with progressive leukodystrophy, non-specific encephalopathy, cardiomyopathy, myopathy, liver disease, Leigh syndrome, Leber hereditary optic neuropathy, and some forms of Parkinson disease.
  • Sequence similarities
    Belongs to the complex I 75 kDa subunit family.
    Contains 1 2Fe-2S ferredoxin-type domain.
  • Cellular localization
    Mitochondrion inner membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • CI-75kD antibody
    • Complex I 75Kd antibody
    • Complex I, mitochondrial respiratory chain, 75 kD subunit antibody
    • Complex I-75kD antibody
    • mitochondrial antibody
    • NADH coenzyme Q reductase antibody
    • NADH dehydrogenase (ubiquinone) FeS protein 1 (75kD) (NADH coenzyme Q reductase) antibody
    • NADH ubiquinone oxidoreductase 75 kDa subunit mitochondrial antibody
    • NADH-ubiquinone oxidoreductase 75 kDa subunit antibody
    • NDUFS1 antibody
    • NDUS1_HUMAN antibody
    • PRO1304 antibody
    see all

Images

  • All lanes : Anti-Ndufs1 antibody (ab52690) at 1/1000 dilution

    Lane 1 : The negative control lane: ~20ug of a total protein extract from E coli ~50ng to 100 ng of a recombinant fusion protein of an irrelevant antigen.
    Lane 2 : Test lane: ~20ug of a total protein extract from E coli with ~50ng to 500ng of the antigen (recombinant fusion protein).

    Secondary
    All lanes : Rabbit anti-mouse IgG + IgM, (H+L) horseradish peroxidase conjugated at 1/5000 dilution

    Predicted band size: 79 kDa
    Observed band size: 32.2 kDa
    why is the actual band size different from the predicted?



    Note: the molecular weight of the band on the western blot does not correspond to the molecular weight of the natural protein because only a fragment of the gene is used.

References

This product has been referenced in:
  • Huai J  et al. TNFa-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation. J Cell Sci 126:4015-25 (2013). Read more (PubMed: 23788428) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for your kind words and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 1123456 and should reach you as requested on the 21.09.12.


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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Answer

Thank you for your reply.

I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful so far. As discussed in my previous email, although other antibodies in your laboratory have worked well under these conditions, all individual antibodies will require individual optimisation. It is not unusual to have to do a few optimisation experiments with a new antibody. We often find that suggesting some scientifically thought out optimisation tips improves the results. This is obviously a preferable outcome for the customer.

However, having revisited the datasheet, it looks like this antibody has not been tested on the endogenous mouse protein so far. I fear that this may be the cause of the problem.I am therefore happy toissue you the replacement with the ab96428 Anti-Ndufs1 antibody.

Usually, our replacements are also covered by our Abpromise, but ab96428is only to predicted to work with mouse samples, and that would mean in case of a failure the experiments are not covered. Checking our website, we have another antibody which is tested to work in mouse: ab104246. Considering the trouble you had with the previous product, I will ask the lab if they can provide a WB image for the mouse lysate to be on the safe side.

Please let me know if I should still send you the replacement of ab96428now or if you would prefer to wait for the answer of the ab104246case.

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Question

Please see attached file. At the moment I am in holiday, when I am back, I shall send you the WB images if absolutely necessary. Best wishes!
Jisen
1) Abcam product code ab52690

2) Abcam order reference number or product batch number 666743
3) Description of the problem
It didn´t work at all, we found many nonspecific bands in our WB,but without specific one.
4) Sample preparation: RIPA buffer lysed
Type of sample (whole cell lysates, fraction, recombinant protein…): whole mouse embryonic fibroblast lysate
Lysis buffer : RIPA buffer
Protease inhibitors: Roche proteinase cocktail
Phosphatase inhibitors: Orthovanadate
Reducing agent: DTT
Boiling for ≥5 min? yes/no Yes
Protein loaded ug/lane or cells/lane: 20µg
Positive control: mitochondria enrichment
Negative control: no
5) Percentage of gel: 10%
Type of membrane: NC
Protein transfer verified: Yes, very well
Blocking agent and concentration: 10% non-fat milk PBST
Blocking time: 4°C overnight+ 1h at room temperature
Blocking temperature: see above
6) Primary antibody (If more than one was used, describe in “additional notes”) : See above
Concentration or dilution : 1/1000 diluted
Diluent buffer: 1%BSA/PBST
Incubation time: 1h, 2h , 3h
Incubation temperature: room temperature
7) Secondary antibody: Donkey anti-mouse HRP
Species: Donkey
Reacts against: Mouse
Concentration or dilution : 1/5000 diluted
Diluent buffer : 1%BSA/PBST
Incubation time: 1h
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer: PBST
Number of washes: 3X5min+3X10min
9)Detection method: ECL
10) How many times have you run this staining? 3 tmes
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? Incubation time

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab52690 Anti-Ndufs1 antibody. I would also appreciate if you can confirm some further details:

1.In order to prevent a masking of your target, please do not block longer than one hour and with not more than 5% milk. Do not over-block. Long blocking incubations, particularly with nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth. 122:129-135, 1989). In addition, omit the Tween from the blocking buffer, as the detergentiswilldecreasethe desired blocking effect.

2. Please keep the blocking agent stringent to one choice, use either milk or BSA. Otherwise it is a bit daunting to find the right blocking agent and to troubleshoot.

3. In order to increase the specificity of the antibody, please incubate over night at 4C. In addition, as you are using a mitochondria fraction, it may be advisable to decrease the antibody concentration further to 1/3000.

4. Can you rule out that the secondary antibody is cross-reacting with your samples?

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information 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.


I look forward to receiving your reply.

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