Overview

  • Product name

    Complex I Immunocapture Kit
    See all Complex I kits
  • Assay type

    Quantitative
  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Optimized for isolation of Complex I from small amounts of tissue for activity assays and other analyses. Based on a highly specific antibody.


    250 µg, 500 µg or 750 µg monoclonal antibody irreversibly crosslinked to protein G-agarose beads which can immunocapture ~60 µg, ~120 µg or ~180 µg respectively of Complex I from heart mitochondria.


    ab109711 allows for isolation of Complex I from small amounts of tissue. This facilitates subsequent analysis of assembly state and activity. Thus the enzyme retains NADH-ferricyanide, NADH-CoQ1 and NADH hexaaminoruthenium reductase activities after isolation, and with added lipids it also shows significant rotenone sensitivity. Finally, the extent of post translational modifications including oxidative damage can be readily analyzed by proteomic approaches or antibody detection of these modifications. Uses for ab109711 include but are not limited to examining alterations of Complex I subunits in inherited mitochondrial diseases, Parkinson's disease, Alzheimer's disease, ALS, schizophrenia, and aging.


    Note: The immunocapture protocol for this kit requires Abcam detergent (lauryl maltoside, ab109857).


    Product supplied as a 6.25% slurry. For the 250 µg, this consists of 250 µg antibody conjugated to 25 µL beads in a volume of 250 µL.

  • 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.

Properties

Images

  • Complex I was immunopurified from mitochondria isolated from human heart (HHM), cow/bovine heart (BHM), mouse heart (MHM) and mouse brain (MBM). The lanes were stained with Coomassie Brilliant Blue R. Bands were excised from the gel and proteolytically digested for mass spectrometry analysis.”

Protocols

References

This product has been referenced in:

  • Milanese C  et al. Mitochondrial Complex I Reversible S-Nitrosation Improves Bioenergetics and Is Protective in Parkinson's Disease. Antioxid Redox Signal 28:44-61 (2018). Read more (PubMed: 28816057) »
  • Bridges HR  et al. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I. Biochim Biophys Acta 1858:197-207 (2017). Read more (PubMed: 27940020) »
See all 17 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Answer

Hello! Below is some more information about kit sizes. Please let me know if I can be of further assistance.

The kits size is based on antibody amount not the beads amount. 250ug refers to 25ul beads, 500ug to 50ul beads and 750ug to 75ul beads. According to the protocol, we recommend to use 10ul beads of each sample, this amount can go down to 5ul but should expect difficulty in handling such small amount of beads. We suggest to use 0.5-2mg materials for each 10ul beads, but should follow the detail direction in the protocol. The yield of complex amount depends on the starting materials, purified mitochondria gives higher yield while cultured whole cell extracts give much lower yield when using same amount of beads and materials.

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Answer


The lab let me know that both ab109711 and ab109798 are IP antibodies - unfortunately they do not work in WB. One can use it to pull down the whole complex I but needs to use other complex I subunit antibodies to blot the corresponding targets.

One would need to buy a series of complex I antibodies to make a cocktail, or just purchase single antibodies to specific subunits of the complex I.


As for the agarose beads: We do not carry them separately, unfortunately.

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Question
Answer

To prepare 10% lauryl maltoside as the detergent for ab109711, pure water (deionized, nanopure etc) can be used. The lauryl maltoside should go into solution fairly easily. Gently turn in rotator, try not to shake it too much to prevent frothing.

I hope this information is helpful to you. Please do not hesitate to contact us with any further questions.

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Question
Answer



In general, for well-prepped mitochondria, the ratio is 1-2 mg of mitochondria per 10 uL of beads. The optimal conditions will depend on the quality and source of the mitochondria so it’s very difficult to be specific, however. A good quality preparation of mammalian heart mitochondria generally only requires 1mg. Other species and tissues may require more so we suggest 1-2 mg per 10 uL of beads. When using cultured cell extract, we recommend 6-15 mg of extract per 10 uL of beads.

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Answer

Hello! Please find the answers to your questions below.

1-elution buffer composition that you recommend in the kit such as 1% SDS.
Is it a buffer or just a water based solution where SDS is solubilized..



It is a water based solution.


2-Second to run a gel you deffinitely can not run the entire volume that you suggest to use for elution. I will starting from 1 mg mitochondria extract
and I will be eluting in 50 ul SDS 1% and I can not load on my gel more than 32 ul. Is that enough to detect proteins by immunoblotting?



We usually load half of the eluted volume on a gel, which is 25 ul/lane. If you start with 1mg mitochondria, complex I should be visualized by Coomassie stain. Immunoblotting is much more sensitive than Coomassie stain. You should expect a fair -strong signal depends on the target you are interested in.

3. We have ab109798 to confirm from the kit.

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Question
Answer

Thank you for contacting Abcam regarding A109711.

I have been in contact with the lab regarding your experiences with this product. However as you have tried a number of the troubleshooting steps already they have requested that I ask you for some additional details.

Could you please tell me what species type you are using this product with?

Do you have any images that you would be willing to share with us?

Are you attempting use of the product with western blot or are you staining the gel? If staining, which product (coomassie, silver stain) are you using to visualize the bands? Could you provide details of your protocols used?

Any information that you would like to include would be of great help in creating the most complete picture for the lab and would be greatly appreciated.

Please let me know if you have any questions or there are other ways in which I might help.

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Answer

Thank you for contacting us.



I have contacted our mitochondria-experts in the lab and received the following reply:

We don’t have a lot of experience with adipose tissue. My suggestion would be therefore to isolate the primary adipocytes from the fat tissue prior to make a mitochondrial preparation.

We have another kit to isolate mitochondria from cultured cells in the catalog: ab110171.(https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html ). This kit could be used for isolating mitochondria from any cell. Preparing the sample in this way, will probably yield the cleanest results with kit ab109711.

Below is a protocolthat you could use to isolate primary adypocytes from the fat tissue.

Primary fat cell isolation.
Fat pads were weighed and cut into 0.3-cm3 pieces with scissors and then were resuspended in KRH, pH 7.4, plus 2.5% BSA containing 1 mg/ml collagenase. The fat pads were digested for 30–45 minutes at 37°C in an orbital bath shaking at 100 rpm. The digested samples were squeezed through chiffon material and the isolated cells were washed 3 times in KRH buffer, pH 7.4, plus 2.5% BSA.

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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Answer

Thank you for your enquiry.

The three different elution methods are recommended to different purposes. Proteins eluted with Glycine remain activity, which is suitable for follow up activity assays. Plus the antibody beads can still be resued. Proteins eluted with urea are compatible to follow up using 2D electrophoresis. Certainly the SDS elution method gives the highest yield of proteins, but the SDS will denature the antibody and it will no longer be viable for use.

To neutralize the glycine, add SDS-PAGE buffer. The bromophenol blue will turn yellow. Add a few uL of 1M Tris pH 7.5 or 100mM NaHCO3 to neutralize the sample and the blue color will return.

I hope this information will be helpful.

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Answer

Thank you for your enquiry. Please find the answers to your questions below.

1) Thank you for your feedback. We will take this into consideration with the production team.

2) Typically customers have used this kit in combination with Complex I activity assays published in the literature. However if you would like to detect rotenone sensitivity, then this ab109903 MitoTox™ Complex I OXPHOS Activity Microplate Assay would be more appropriate. It is a plate based assay that contains lipids and is rotenone sensitive. However, the lipid content of this kit is proprietary.

I have attached a paper showing how to perform activity assay after immunocapture with the beads. This protocol uses BSA instead of the lipid (see figure 2 under results).

A Functionally Active Human F1F0 ATPase Can be Purified by Immunocapture from Heart Tissue and Fibroblast Cell Lines. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 37, pp. 33906–33912, 2002

I hope this information will be helpful.

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Answer

The MitoSciences laboratory recommends the following: Use 1 mg of brain tissue lysate, eluting into the minimum volume possible. A glycine buffer elution procedure allows the reuse of the beads. If you wish to analyze the product by Western blot, we can scale back the amount of material required. So using whole tissue I think you can be very confident using 1 mg of tissue or less. However, be aware of possible mouse capture antibody elution from the beads into the sample when blotting. Ways to resolve this problem: (1) Do not include reducing agent in sample during electrophoresis. This should maintain eluted antibody at 200 kDa during electrophoresis. (2) Western blotting – Blot with rabbit antibodies or use a secondary detection system that recognizes only the native mouse antibody being used to Western blot.

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1-10 of 12 Abreviews or Q&A

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