Total OXPHOS Human WB Antibody Cocktail (ab110411)

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.

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.

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.

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

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

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.

Thank you for your inquiry.

There is a representative protein from each of the 5 OXPHOS complexes in this mouse monoclonal antibody cocktail (ab110411, MS601).

I can confirm that one protein, COX-II (Uniprot P00403) is mtDNA encoded and all the others are nuclear encoded.

There is another OXPHOS cocktail ostensibly labeled for Rodents, which will also work on human samples (MS604 ab110413)

Again one protein, in this case COX-I (uniprot P00395), is mtDNA encoded and the others are all nuclear encoded.

I hope this information is helpful and wish you good luck for your experiments.

Hello! A few more tips for troubleshooting!

Blue native (BNPAGE) doesn’t have great power of resolution (unlike SDSPAGE),t he main reason for this is that the complexes are in multiple states of assembly therefore some give multiple and often fuzzy bands.

The only way to do this kind of blotting with a cocktail is to take the BNPAGE gel and rotate it through 90 degrees and run it in a second, SDSPAGE, dimension then blot.

Using this cocktail or we have a specific blue native cocktail, we have a specific product for this and show how it is done on the associated datasheet:

https://www.abcam.com/MitoProfile-Total-OXPHOS-Blue-Native-WB-Antibody-Cocktail-ab110412.html

Experimental details below in our protocol.



There is a second option where the first dimension can be blotted, but in this approach only one antibody at a time can be used this will lead to a series of blots with fewer bands which are hopefully more easy to interpret.

Single antibodies compatible with first dimension NBPAGE are

MS111 Complex I subunit NDUFA9 monoclonal antibody

MS105 Complex I subunit NDUFB8 monoclonal antibody

MS103 Complex I subunit GRIM-19 monoclonal antibody

MS204 Complex II subunit 70 kDa Fp monoclonal antibody

MS304 Complex III subunit Core 2 monoclonal antibody

MS404 Complex IV subunit I monoclonal antibody

MS507 ATP synthase (Complex V) subunit alpha monoclonal antibody





Details for doing BNPAGE and blotting in one and two dimensions are given in our protocol:

http://www.mitosciences.com/PDF/BNPAGE.pdf

Thank you for contacting Abcam. I am sorryyou are experiencingdifficulty with this antibody cocktail. I am wondering whether you have tried somthing simple like reducing the primary incubation time. Based onyour image it seems that you couldreduce the primary incubation to 2 hr at room temperature. Please let me know if this is something you have already tried.

Thank you for contacting Abcam regarding ab110411.


More than likely the antibodies in the cocktail will be okay and function as expected. The product is shipped on dry ice and sometimes arrives frozen and this has not impacted product quality. I would recommend storing at the recommended temperature and avoid any more freeze/thaw cycles. If any damage has been done, you may observe this in the form of needing more antibody to get a signal or the potential for some background, but again, this is unlikely.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.