Total OXPHOS Blue Native WB Antibody Cocktail (ab110412)

The antibody concentrations in ab110412Total OXPHOS Blue Native WB Antibody Cocktail are only optimized for the example provided on the datasheet.  Maximum signal, minimal background for each protein in cultured cells e.g. fibroblasts.

 
The challenge with antibody cocktail signals is that they are sample and tissue dependent.  In some samples there will be signal parity but not in others. This may require individual optimization with multiple exposures and composite analysis depending on the sample.

Both of these cocktails measure a representative subunit from each of the 5 OXPHOS complexes. Both work on human, mouse and rat.

The MS603 was designed first and is for use when Western blotting a 2D BNPAGE gel to get a good separation of the complexes in the second dimension.
When doing 2D BNPAGE and comparing normal and experimental samples, a change in the spot intensity OR a shift in the position an OXPHOS complex (to the right as shown in the figure) would indicate that the assembly of the enzyme is affected.
This is a complex experiment in terms of sample handling but also assembly of equipment.
MS604 on the other hand is simpler, it measures a representative protein in a regular 1D SDS PAGE Western blot. A change in the level of that protein can be indicative of changes in assembly. However it’s possible that assembly changes can happen without affecting the level of this representative protein. We do find that in many cases the levels do change as shown by the lysate control that is available-
https://www.abcam.com/Hela-Rho0-treated-and-control-Whole-Cell-Lysate-ab154479.html
Note the data is shown for MS601 (the human-only version of MS604) but the results would be very similar.

Vielen Dank für die Zusammenfassung in Englisch. Ich habe nun eine Antwort von unseren Mitochondrien -Spezialisten aus dem Labor erhalten.

Ich habe die Antwort nicht übersetzt um Übersetzungsfehler zu vermeiden:

"Let me start by saying these are very good questions and extremely difficult to answer as well. Here are the answers to your questions

Buffy coat samples to extract PBMC are available that have been stored at -196°C since the blood draw 20 years age. Are these samples appropriate for your assays?



It depends on the buffer in which they have been stored. I’m assuming from the temperature specification that the samples have been frozen in the vapor phase of liquid nitrogen. If they were frozen in freezing media containing 30 – 50% FBS + 10% DMSO + Standard Culture media, then they will be viable and in good condition to determine protein based end point biomarkers. If this is not the case, it will be difficult to get good proteomic data out of the samples because the freeze/thaw process may break the granulocytes and release proteases that will completely degrade the samples before you have a chance to work with them. These proteases are extremely potent and are difficult to inhibit with regular protease inhibitors available in the market.



Do you have any idea which markers would make sense in the context of Alzheimer’s disease (e.g. OXPHOS complexes)?



We have a fair amount of experience with Mitochondria, however our experience with the use of PBCSs as surrogate markers of disease states is more limited. We only have experience of this in the field of HIV and retroviral therapy, where OXPHOS proteins (CI and CIV) have been shown to be potential good surrogate markers of therapy toxicity in this subgroup of patients. Do you want to use PBMCs as a surrogate tissue for the discovery or for the validation of biomarkers of brain injury (Alzheimer’s Disease)?. If you are planning a DISCOVERY type of experiment, my suggestion is to look more broadly in the mitochondria proteome and even in the whole cellular proteome. Antibodies and Immunoassays are not very suited for this type of discovery research. There is a paper in the literature where they used a combined methodology (genomic/transcriptomic/proteomic profiling) to discover Alzheimer’s disease markers in the blood. This was published by a group at the University of Louisville Email: mailto:eugenia.wang@louisville.edu. My suggestion is to email the senior author to discuss their results and potentially collaborate with them to select true candidate targets for validation.



Antibodies and Immunoassays are particularly useful in the validation stage, where you have selected a number of targets in the discovery phase and want to validate the results (remember that discovery phases have a large number of false positives even when this is controlled statistically with the use of “false discovery rate”). If you find in your talks with Professor Wang that the OXPHOS complexes or other mitochondria targets are in the list of candidate biomarkers, then the questions is how to use the best product/platform that measures that target, which is your next question.



Which technique would you suggest to use for a large-scale study? We are working on a tight budget





The answer to this question depends first on your accessibility to instrumentation. Do you have access to plate readers, are they colorimetric or fluorescent?. Do you have access to a flow cytometer? How many people will perform these experiments (data on 6000 samples with the use of immunoassays is not trivial), are they located in a single centralized large lab or in multiple labs around the world?



We have a platform called “Dipsticks” or lateral flow immunoassay which have been used by previous researchers in the past with PBMCs on other types of diseases. I can provide references with the use of Dipsticks in PBMCs in mitochondrial disease, frataxin, HIV/antiretroviral therapy and traumatic brain injury (which used PDH as a marker). These studies were a lot smaller than yours though. The main disadvantage of Dipsticks is that they are not amenable for high throughput. However researchers have used them in these studies, because they are extremely easy to use and require minimal training, for quantitative data they require a specialized reader “dipstick reader” which costs a fraction of what a regular microplate reader costs and could be easier to use in the future as potential “point of care research tests”. Bear in mind that all our products are RUO (research use only). Dipsticks are inexpensive but do require a large amount of “man power” and many of them have a higher sensitivity than the microplate counterpart. Our dipsticks measure the target proteins either in a sandwich ELISA or with an activity assay. Your other option could be microplates, which will allow you to run 96 tests at a time. I discourage you from using western blot in so many samples. Western blot is not quantitative when it comes to comparing results from one blot to the next, whereas the microplates or dipsticks are easier to compare from run to run. I encourage you to visit our metabolism page and look at different targets in these two platforms. On the main page there is a video about the dipsticks which will allow you to learn more about them. Once you decide on a particular target/platform we will happily help you with further scientific support.


https://www.abcam.com/metabolism/



The other page you should visit is our ELISA and activity assay kits page (below) for dipsticks and microplates



https://www.abcam.com/index.html?pageconfig=resource&rid=13855



https://www.abcam.com/index.html?pageconfig=resource&rid=13918"



Ich hoffe diese Information ist hilfreich und wünsche Ihnen viel Erfolg mit Ihrer Studie.

Thank you for contacting us.

In theory, any antibody can be used in the “second” dimension of the Blue Native electrophoresis. In the first dimension, proteins are in their native form and only antibodies validated to work in this platform will work. That is why we have a cocktail designed to detect the complexes by BN PAGE in the first dimension (ab110412 / MS603). Once the proteins from the complexes are resolved in the second dimension (denatured dimension), then any WB positive should work, including the antibodies in ab110411 / MS601.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for contacting us. As you may have seen, there is an image of a 2D denatured gel on the product datasheet. Unfortunately, we have an image of a denatured gel not available, and we have not used this product for blotting the 1D native gel because not all antibodies would work in the native conditions and the signals would be hard to discern from one another in the 1D. Therefore we do a second dimension to resolve spots in an extra dimension. However, if you would like to do first dimension native blotting, we strongly recommend doing individual strips for complex-specific blotting using native binding antibodies, which are listed on our protocol in addition to the non-native protocol (ab14713-ab14748, attached). Usually, it is recommended to do blue native gel electrophoresis on the mitochondria fraction isolated from the cells rather than using whole tissue homogenates, because clean mitochondria should not contain IgG. However, if there are issues we often recommend a secondary HRP-conjugate which binds only the native detector antibody cocktail and therefore prevents mouse on mouse issues. We have for example antibodies available that do not detect the IgG heavy chain, and I would be pleased to assist you in finding the most suitable product if requested. Otherwise, I would like to recommend checking the Biocompare website which has an excellent antibody search facility that includes many suppliers ( http://www.biocompare.com ). I hope this information is helpful to you. Please do not hesitate to contact me if you have any further questions in this regard.

Thank you for contacting us. The Blue Native Western Blot does not require specific gels. Native acrylamide gels can be poured by hand. While it is possible to use a single acrylamide concentration we recommend the use of a linear acrylamide concentration. For more details about Blue Native Western Blotting, I suggest to look at the protocol available via this link : http://www.mitosciences.com/PDF/BNPAGE.pdf 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 call today and for your questions about ab110412 (MS603). I have found that this antibody is suitable for use after 2-D separation. We do have some data at the bottom of the datasheet that shows staining following 2-D separation- https://www.abcam.com/mitoprofile-total-oxphos-blue-native-wb-antibody-cocktail-ab110412.html I hope this information is helpful, but please let me know if you have any questions or if there is anything else we can help with.