Overview

  • Product name

  • Description

    Rabbit polyclonal to Ndufs1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WBmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Cow, Zebrafish
  • Immunogen

    Recombinant fragment, corresponding to amino acids 1-162 of Human Ndufs1

  • Positive control

    • MOLT4 whole cell lysates

Properties

Applications

Our Abpromise guarantee covers the use of ab96428 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. Predicted molecular weight: 79 kDa.

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

  • Anti-Ndufs1 antibody (ab96428) at 1/1000 dilution + MOLT4 whole cell lysate at 30 µg

    Predicted band size: 79 kDa



    12% SDS PAGE

References

This product has been referenced in:

  • Jacquemin G  et al. Granzyme B-induced mitochondrial ROS are required for apoptosis. Cell Death Differ 22:862-74 (2015). Read more (PubMed: 25361078) »
  • Tun AW  et al. Profiling the mitochondrial proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: down-regulation of bioenergetics and mitochondrial protein quality control pathways in fibroblasts with the 11778G>A mutation. PLoS One 9:e106779 (2014). WB ; Human . Read more (PubMed: 25215595) »
See all 2 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

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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