Pyruvate dehydrogenase (PDH) WB Antibody Cocktail (ab110416)

Thank you for contacting us.

Please find the WB protocol used to validate our MitoSciences range of antibodies attached, as well as the pdf datasheets for those products. Keep in mind, we regularly update our online datasheets to reflect the most recent information including additional protocol notes, images, testing new species and applications, and customer feedback.

The recommended concentrations for these antibodies are as follows:

ab110416 (MSP02) MitoProfile Pyruvate dehydrogenase (PDH) WB antibody cocktail: 6 ug/mL
ab110330 (MSP03) anti-Pyruvate Dehydrogenase E1-alpha subunit antibody: 1 ug/mL
ab110282 (MS703) anti-3 Nitrotyrosine antibody: 1 ug/mL
ab110413 (MS604) MitoProfile Total OXPHOS Rodent WB antibody cocktail: 6 ug/mL

While we used Amersham ECL+ for validation of these antibodies, our new Optiblot ECL Max Detect Kit will give a very strong signal with low background, and is suitable for detecting 4.6pg - 4.7ng of protein (ab133408).

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

Thank you for contacting us regarding the use of ourMitoProfile® Pyruvate dehydrogenase (PDH) WB Antibody Cocktail in rat tissues.

I have gathered the following information regarding each antibodies reactivty with rat below:



Cocktail Antibodies:



Mouse anti C-V-OSCP [4C11C10D12] monoclonal: This has not been tested in rat, alignment of rat to human and bovine is very poor, we do not expect this to work



Mouse anti PDH-E1-alpha [9H9AF5] monoclonal: has been successfully tested in rat



Mouse anti PDH-E1-beta [17A5E2H8] monoclonal: has been successfully tested in rat



Mouse anti PDH-E2 [15D3G9C11] monoclonal: has not been tested in rat, alignment shows a 85% homology, we would expect this product to work in rat



Mouse anti PDH-E2/E3bp [13G2AE2BH5] monoclonal: has been successfully tested in rat



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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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 your inquiry.

I am happy to confirm the molecular weight of the following proteins:

O (complex 5) : 22kD

E1beta : 39.4kD

E1alpha : 43.3kD

E3bp : 54kD

E2 : 69kD

I also attached the image including the size marker to this email. The image on the datasheet will be up-dated as soon as possible.

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

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.