Total OXPHOS Human WB Antibody Cocktail (ab110411)

Reviews (4)Q&A (23)References (431)
Western Blot Abreview Image
  • Western blot - MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411)

Key features and details

  • Assay type: Quantitative

Overview

  • Product name

    Total OXPHOS Human WB Antibody Cocktail

  • Assay type

    Quantitative

  • Species reactivity

    Reacts with: Human

  • Product overview

    ab110411  contains 5 mAbs, one each against Complex I subunit NDUFB8 (ab110242), Complex II subunit 30kDa (ab14714), Complex III subunit Core 2 (ab14745), Complex IV subunit II (ab110258), and ATP synthase subunit alpha (ab14748) as an optimized premixed cocktail. The cocktail is suitable for Western Blotting analysis of the relative levels of the 5 OXPHOS complexes in mitochondrial preparations from human sources.


    Altered levels of assembly can arise from mutations in individual subunits, mutations in assembly factors for the complex(es), mtDNA depletion or as a result of physiological and or pathological changes e.g. hormone treatment, exercise, diet or oxidative stress.


    The mAbs in the cocktail were chosen because they are against a subunit that is labile when its complex is not assembled. Moreover, the combination is readily resolved in SDS-PAGE when the appropriate gel conditions are used (Please refer to application notes).


    The Western blot cocktail is supplied at a concentration of 1.5 mg/mL.


    The five antibodies provided in the cocktail are all mouse monoclonal antibodies.


    We recommend using a high pH CAPS buffer / PVDF transfer protocol when using this antibody for Western blot. Please contact us for more details.


    Due to a policy change, the previously provided control for this kit has been removed.


    We do not recommend to boiling samples since it will cause signal reduction of several bands. We recommend warming to 37°C for 30 minutes prior to loading.

  • Notes

    Related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Tested applications

    Suitable for: WBmore details

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab110411 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
Use at an assay dependent concentration.

Recommended starting dilutions is 1:200 - 1:1000. However, optimal diltuion should be established by the end-user.

We recommend using a high pH CAPS / PVDF transfer protocol when using this antibody for Western blot. Please contact us for more details.

We do not recommend to boiling samples since it will cause signal reduction of several bands. We recommend warming to 37°C for 30 minutes prior to loading.
Notes
WB
Use at an assay dependent concentration.

Recommended starting dilutions is 1:200 - 1:1000. However, optimal diltuion should be established by the end-user.

We recommend using a high pH CAPS / PVDF transfer protocol when using this antibody for Western blot. Please contact us for more details.

We do not recommend to boiling samples since it will cause signal reduction of several bands. We recommend warming to 37°C for 30 minutes prior to loading.

Images

  • Western Blot Abreview Image
    Western Blot Abreview ImageThis image is courtesy of an anonymous Abreview

    Gel: Non-reduced Denaturing (4-12%)

    Lanes 1-3: contain 3 individual Human B cell lymphoma samples 50 μg loaded per lane.

    Blocked for 1 hour with 5% Milk at room temperature.

    ab110411 used as per instructions.

    Secondary: HRP-conjugated anti-mouse IgG uesd at 1/300

    Detection method: ECL+ 2 minutes exposure.

     

  • Western blot - MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411)
    Western blot - MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411)
    Each lane contains 10 µg of mitochondria isolated from MRC5 fibroblasts.
    ab110411(MS601) was used to label lane 6, and the individual antibodies
    comprising the cocktail were used for lanes 1-5.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (431)

Publishing research using ab110411? Please let us know so that we can cite the reference in this datasheet.

ab110411 has been referenced in 431 publications.

  • van Beek SMM  et al. Effect of β2-agonist treatment on insulin-stimulated peripheral glucose disposal in healthy men in a randomised placebo-controlled trial. Nat Commun 14:173 (2023). PubMed: 36635304
  • Hao X  et al. Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress. Oncogene 41:427-443 (2022). PubMed: 34773075
  • Mennuni M  et al. Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen. EMBO Rep 23:e53054 (2022). PubMed: 34779571
  • Minnock D  et al. Altered muscle mitochondrial, inflammatory and trophic markers, and reduced exercise training adaptations in type 1 diabetes. J Physiol 600:1405-1418 (2022). PubMed: 34995365
  • Van Bergen NJ  et al. Biallelic Variants in PYROXD2 Cause a Severe Infantile Metabolic Disorder Affecting Mitochondrial Function. Int J Mol Sci 23:N/A (2022). PubMed: 35055180
View all Publications for this product

Customer reviews and Q&As

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1-10 of 27 Abreviews or Q&A

Western

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
MitoProfile Total OXPHOS
40 ug total protein from human heart tissue whole lysate
12% tris-glycine gel
5% milk-TBST block, 1 h, RT
primary of 1:750 in 5% milk for 16 h at 4C
secondary of 1:1250 in 5% milk for 1 h at RT
exposure varied based on band
excellent ease of use - 5/5
image attached
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 Oct 29 2014

Total OXPHOS Antibody for Western Blotting

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
20 ug total protein from neuroblastoma cell line
13.5% SDS-PAGE
3% milk-TBST block, 1 h, RT
primary antibody 1:1000 in 3% milk-TBST for 16 h at 4C
secondary antibody 1:1000 in 3% milk-TBST for 2 h at RT
exposure varied (10 seconds to 1 minute) based on band
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 Dec 24 2018

Total OXPHOS antibody for western blotting

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
human fibroblast whole lysate used to detect OXPHOS
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 Jul 10 2017

Total OXPHOS antibody cocktail

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
In this experiment we used isolated mitochondria from human HCT116 colon cancer cell line. The proteins from the mitochondrial lysate were seperated on a 13% SDS gel. The total OXPHOS human WB antibody kit was diluted 1:1000 in 2% milk. We already obtained strong signals of the complexes V and I after 10 sec. After 1 min (attached figure) we got signals from all five complexes.

The kit is easy to use and gives reproducibly results.
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.

Mr. Christian Marx

Verified customer

Submitted Aug 21 2013

Question

is it necessary this cocktail be used with non-reducing conditions or can I reduce?

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Answer

Thank you for contacting us.

The answer is yes, you can use reducing reagent in the sample but we do not recommend boiling samples since it will cause signal reducing of several bands.

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

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Question

1. what do you mean by labile protein for the differents complexes? are they degraded or found in the cytoplasm?
2. Is a mitochondrial extraction necessary?
3. Is there a pharmacological treatment that you would recomand as a positive control to monitor complex deficiency? eg: rotenon, oligomycin?

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Answer

Thank you for your enquiry.

I am pleased to help answer your questions:

1. what do you mean by labile protein for the differents complexes? are they degraded or found in the cytoplasm?

Several of the proteins are degraded, some more rapidly than others. We recently made a lysate product to compliment this product – a lysate of rho0 cells (lacking mitochondrial DNA), and the loss of these proteins as they are degraded can be observed.
Hela-Rho0 treated and control Whole Cell Lysate (ab154479)
https://www.abcam.com/HeLa-Mitochondrial-DNA-Depletion-Cell-Lysate-Set-Ethidium-Bromide-Treated-and-Vehicle-Treated-ab154479.html (or use the following: https://www.abcam.com/HeLa-Mitochondrial-DNA-Depletion-Cell-Lysate-Set-Ethidium-Bromide-Treated-and-Vehicle-Treated-ab154479.html).

2. Is a mitochondrial extraction necessary?
No, whole cell lysate using SDS-PAGE western blot buffer will be good enough.


3. Is there a pharmacological treatment that you would recomand as a positive control to monitor complex deficiency? eg: rotenon, oligomycin,....?
An agent to remove mtDNA would be useful. For example EtBr, DDC or to prevent mtDNA expressed protein translation such as chloramphenicol and several other antibiotics.

These agents act over several cell doublings resulting in the removal of mtDNA encoded proteins which are degraded. The inhibitors you mention may be a bit potent – inhibiting the enzymes first before an effect is seen on protein expression/turnover.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact me.

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Question

Dear Sir/Madam,

I was trying to get a datasheet for the product MS602 but I failed on your homepage and the one of Mitoscience.

Can you please send me the datasheet.

In fact, what I would need is an OXphos/PHD human AK kit for IHC and WB, if possible!
Kind regards

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Answer

Thank you for your enquiry. It has taken some time to confirm all the details for you, and I appreciate your patience.

I am sorry to confirm that MS602 has been discontinued and we do not have anymore in stock.

Although that product is not available any longer, there is ab110411, Total OXPHOS Human WB Antibody Cocktail and ab110416, Pyruvate dehydrogenase (PDH) WB Antibody Cocktail, however as the name suggests they have only been tested in WB, not IHC-P.

If you would like to considering trying to recreate the original antibody cocktail,I have listed the individual components are below.

Complex I 17G3D9E12 ab110243
https://www.abcam.com/NDUFB4-antibody-17G3D9E12-ab110243.html (or use the following: https://www.abcam.com/NDUFB4-antibody-17G3D9E12-ab110243.html).

Complex II ab14714
https://www.abcam.com/SDHB-antibody-21A11AE7-ab14714.html (or use the following: https://www.abcam.com/SDHB-antibody-21A11AE7-ab14714.html).

Complex III 13G12AF12BB11 ab14745
https://www.abcam.com/UQCRC2-antibody-13G12AF12BB11-ab14745.html (or use the following: https://www.abcam.com/UQCRC2-antibody-13G12AF12BB11-ab14745.html).

Complex IV 1D6E1A8 ab14705
https://www.abcam.com/MTCO1-antibody-1D6E1A8-ab14705.html (or use the following: https://www.abcam.com/MTCO1-antibody-1D6E1A8-ab14705.html).

Complex V 4C11C10D12 ab110276
https://www.abcam.com/ATP5O-antibody-4C11C10D12-ab110276.html (or use the following: https://www.abcam.com/ATP5O-antibody-4C11C10D12-ab110276.html).

PDH 9H9AF5 ab110330
https://www.abcam.com/PDHA1-antibody-9H9AF5-ab110330.html (or use the following: https://www.abcam.com/PDHA1-antibody-9H9AF5-ab110330.html).

As there are several components, we would be able to offer you a discount if you wish to purchase all of these products together. I am forwarding this to our bulk orders team who will be able to provide a quote for you.

Otherwise, I am sorry there is nothing else to suggest that would be a suitable replacement on this occasion.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

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Question

Are all the subunits analyzed encoded by nuclear DNA? If not, could you specified which are encoded by nuclear or mtDNA? thank you

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Answer



The link below shows the genes in the mitochondrial DNA. This is a very comprehensive review.

http://www.nature.com/nrg/journal/v13/n12/full/nrg3275.html

There are 37 mitochondrial DNA (mtDNA)- encoded genes which include seven subunits of complex I (ND1, 2, 3, 4, 4L, 5 and 6), one subunit of complex III (cytochrome b (Cyt b)), three subunits of complex IV (Cyt c oxidase (COX) I, II and III), two subunits of complex V (A6 and A8), two rRNAs (12S and 16S) and 22 tRNAs (one-letter code).

For product ab110411, proteins are as follows:

Complex I = NDUFB8 – Nuclear encoded

Complex II = SDHB – Nuclear encoded

Complex III = UQCRC2 – Nuclear encoded

Complex IV = MTCO2 – Mitochondrial encoded

ATPase = ATP5A – Nuclear encoded

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Question

Have you or anyone tested this mixture on whole tissue or whole cell homogenate, not isolated mitochondria?

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Answer


In-house we test this cocktail using isolated mitochondra, but other users have published data using whole cell or whole tissue homogenates. We have these publications listed on the datasheet-

https://www.abcam.com/MitoProfilereg-Total-OXPHOS-Human-WB-Antibody-Cocktail-ab110411-references.html

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Question

So, if I am correct, the Complex II (mtDNA encoded COX2---> MT-CO2) from the anti-body cocktail is actually part of complex IV of the oxidative phosphorylation (OXPHOS). This makes the description of the anti-body cocktail a bit confusing(especially when you talk about complex...). I got confused because Complex II components of OXPHOS are not encoded by mtDNA, they are all nuclear encoded.



bw,

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Answer

Thank you for your reply.

I can understand why you find the nomenclature a bit confusing and we will certainly strive in the future to make the nomenclature on our datasheets more consistent by ensuring that the full name is added to the target alongside the acronym.



However, the current nomenclature is the standard in the field. COX is the short name for Cytochrome c Oxidase and not for Complex. If you look in the Uniprot page the recommended name is Cytochrome c oxidase subunit 2 with the following acronyms (MT-CO2, COII, COXII, MTCO2). On the other hand Complex II is denoted in the field as SDH (Succinate dehydrogenase). In Uniprot you will find the different subunits as SDH followed by a letter or number to denote the subunit number.



See Uniprot



P00403 (COX2_HUMAN) - http://www.uniprot.org/uniprot/P00403




Name:
MT-CO2

Synonyms:
COII, COXII, MTCO2




Recommended name:
Cytochrome c oxidase subunit 2



P31040 (DHSA_HUMAN) - http://www.uniprot.org/uniprot/P31040




Name:
SDHA

Synonyms:
SDH2, SDHF




Recommended name:
Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial

I hope this information is helpful and wish you a good weekend.

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1-10 of 27 Abreviews or Q&A

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