Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer) (ab110171)

Reviews (1)Q&A (25)References (19)
Western blot - Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer) (ab110171)

    Key features and details

    • Assay type: Quantitative

    Overview

    • Product name

      Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer)
      See all Mitochondria Isolation kits

    • Assay type

      Quantitative

    • Species reactivity

      Reacts with: Mammals, Other species

    • Product overview

      ab110171 contains sufficient reagents for up to 20 benchtop mitochondria isolations from mammalian cultured cells. Isolated mitochondria can be analyzed by Western Blotting, Immunocapture, or Activity assay.

       

       

    Properties

    Images

    • Western blot - Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer) (ab110171)
      Western blot - Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer) (ab110171)
      Isolated mitochondria show enriched signal when compared to the whole cell extract. In lane 1, MRC5 mitochondria isolated with Abcam's ab110171/MS853 Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer) were loaded at 20 µg. In lanes 2 and 3, post-spin supernatant (SN) and whole cell extract were loaded at 20 µg.

    References (19)

    Publishing research using ab110171? Please let us know so that we can cite the reference in this datasheet.

    ab110171 has been referenced in 19 publications.

    • Nakano D  et al. Effects of canagliflozin on growth and metabolic reprograming in hepatocellular carcinoma cells: Multi-omics analysis of metabolomics and absolute quantification proteomics (iMPAQT). PLoS One 15:e0232283 (2020). PubMed: 32343721
    • Takashi Y  et al. Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis. Free Radic Biol Med 161:60-70 (2020). PubMed: 33017631
    • Chen Q  et al. BRCA1 Deficiency Impairs Mitophagy and Promotes Inflammasome Activation and Mammary Tumor Metastasis. Adv Sci (Weinh) 7:1903616 (2020). PubMed: 32195105
    • Oleinik N  et al. Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress. Sci Adv 5:eaax1978 (2019). PubMed: 31535025
    • Wang Y  et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep 29:1511-1523.e5 (2019). PubMed: 31693892
    View all Publications for this product

    Customer reviews and Q&As

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    1-10 of 26 Abreviews or Q&A

    Mitochondrial fractionation kit

     Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    abreview image
    This kit is very easy to use to isolate intact mitochondria from adherent cells. The kit was used for mitochondria isolation from human iPSC-derived midbrain dopaminergic floorplate progenitor cells. Adherent cells can simply be scraped from the plate and subjected to differential centrifugation in the kit provided buffers to generate a pure mitochondrial fraction. In this application, absence of mitochondria from cytosolic fraction was confirmed by subjecting cytosolic and mitochondrial fraction to a WB for the mitochondria-specific antibody TOMM20.

    Abcam user community

    Verified customer

    Submitted Dec 30 2019

    Question


    I am using Mitochondria isolation kit for cultured cells (MS853).
    I want to try immuneprecipitation (IP) assay from mitochondrial fraction.
    I assume this kit can isolate intact mitochondria with complete structure.
    I'm wondering whether the final product can be applied to IP reaction. My concern is that IP reaction might not be efficient with intact mitochondria. Should I lyse the mitochondrial pellets again in order to disrupt mitochondrial structure and get enough amount of mitochondrial matrix protein before applying to IP? If yes, do you have any recommendation for lysis buffer?

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    Answer

    1. Yes, the kit isolates intact mitochondria
    2. We recommend lysing the mitochondria for IP using lauryl maltoside:
    10% lauryl maltoside: ab109857, https://www.abcam.com/10-lauryl-maltoside-solution-ab109857.html
    15% lauryl maltoside: ab109858, https://www.abcam.com/15-lauryl-maltoside-solution-ab109858.html

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    Question

    Customer kindly contacted us wondering if Reagent C could be frozen after adding protease inhibitors for longer lifetime.

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    Answer

    Reagent C of ab110171 can be frozen. This may extend the shelf-life of the reagent, particularly if it has been opened and a bacterial growth is of concern. Since we do not provide the protease inhibitors, we do not know how stable the protease inhibitors are after being diluted. In general, it is recommended to add protease inhibitors just before the use.

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    Question

    Thanks for this thorough answer. Does reagent A contain any protein e.g. BSA?
    Regards,

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    Answer

    Thank you for contacting me. You are welcome with any questions.

    I double checked with the laboratory. Reagent A contains no protein. It is a buffered solution containing sucrose and optimised detergents. Regrettably the exact contents it proprietary information we can not chare with our customers.

    I hope this is of help nevertheless. Please do not hesitate to come back to me if you require more information.

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    Question

    Dear technical team,

    Our customer is going to purchase ab110171 and would like to know information of homogenizer such as material, shape (could you give me picture?), http://endic.naver.com/enkrEntry.nhn?entryId=a633cfcee8eb4eaa835c11c2a9b8996c&query=disposable or reusable etc.
    Please let me know.

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    Answer

    Thank you for contacting us.

    The homogenizer is borosilicate glass and is reusable. Below is a link to a full description and picture:


    https://us.vwr.com/store/catalog/product.jsp?catalog_number=KT885300-0002


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

    Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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    Question

    Customer kindly contacted us inquiring about sample preparation and storage.

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    Answer

    Thank you for contacting us.

    Samples may be prepared as purified mitochondria, crudemitochondria. In most cases whole tissue or cell lysates aresuitable but these may require some sample optimization. Mitochondria samples can be prepared bysimple differential centrifugation of homogenized samples. While any crude isolation may be effective, we recommend ourhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html(with Dounce homogenizer) if using cultured myocytes such as HL-1 cells.If using heart tissues we would recommendhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html(with Dounce homogenizer).



    This kit was optimized to extract complex I into its native form (with all 47 subunits assembled in to a large complex). The sample extraction is native so care needs to be taken to extract the samples at equivalent starting concentrations to maintain the protein: detergent ratio and maintain enzyme integrity and activity.Specifically for this product - the extraction is performed by adding 1/10 volume of the 10X Detergent to samples at 5.5mg/mL



    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

    I'm looking for a lysis buffer condition that will solubilize plasma membrane but leave mitochondria intact - any suggestions? With the buffer that you recommend, what happens to nuclei?

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    Answer

    Thanks for your enquiry.

    We have two kits that are designed to isolate mitochondria:

    1. ab110168 - Mitochondrial isolation kit for tissues
    2. ab110171 - Mitochondrial isolation kit for cultured cells

    You can check the Protocol booklet links on the online product datasheets for protocol specifics. In general, for both kits, a nuclear pellet is produced by the first spin and a mitochondrial pellet is produced during the second spin.

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    Question

    Hi there,

    I have a customer who has been experiencing difficulties with the Mitochondrial isolation kit (ab110171). She purchased ab110170 and ab110171 in August 2011. Please see the issue below, and attached technical questionnaire

    The main problem I have encountered is that the final pellet contains gooey substance which in indicative of the presence of DNA. I have been adhering to the Abcam protocol and have repeated the isolation a few times to no avail. The DNA contamination effectively renders the pellet unusable as I need a pure mitochondrial pellet product.

    I was completely unaware of the Abcam guarantee which is a shame because I used them within a month of arrival and have not used them since. One of the kits still remains unopened.

    I've attached the completed enquiry form as requested,

    Can you please help with this issue?

    Many thanks,

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    Answer

    Thank you for contacting us and reporting the problems this customer has been having with the Mitochondria Isolation Kit for Cultured Cells kits (ab110170 and ab110171).

    As mentioned, I have consulted with the lab in MitoSciences to get their opinion on this case. They have informed me that the problem encountered by your customer is most likely due to the homogenize step being over-done. In order to resolve this, the customer can either add DNAnase (such as benzonase) to clean up the DNA contamination or repeat the mitochondrial isolation with less and gentlerstrokes since her sample was frozen cells.

    I hope this information has been of help. It is unfortunate that the customer did not contact us earlier in order for us to be able to help her. I'm glad she is now aware of the Abpomise.

    If you have any further questions, please do not hesitate to contact us again.

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    Question

    Can you tell me more about the Dounce homogenizer in this kit?

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    Answer

    Thank you for your enquiry.

    The Dounce Homogenizer in this kit is made of glass and includes 2 pestles, and one 2ml column. I have included an image for your reference.

    If there is anything else that I can help you with please do not hesitate to contact me.

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    Question

    I would like to run the isolated mitochondria on a Western blot. In figure 2 on your datasheet, you show Western blot results with the mitochondrial fraction. Could you tell me how that fraction was prepared for Western blot (i.e. were lysis buffer and sample buffer added)? Is the buffer compatible with a BCA assay?

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    Answer

    Thank you for your call yesterday and for your patience while I have been in touch with the lab regarding ab110171.

    The lab used the following procedure to run the mitochondrial fraction on a Western blot:

    1. Measure the protein concentration using BCA assay orNanoDrop after the mitochondrial fraction preparation.

    2. Solubilize the mitochondrial fraction with SDS to a final concentration of 1% SDS and 5mg/ml proteins.

    3. Dilute the sample with 5X Laemmli buffer and TBS (final concentration is 1X Laemmli buffer, 2mg/ml proteins).

    4. Load 10 to 20 ul sample per lane.

    Since the protein concentration is measured prior to adding the buffers, everything will be compatible with a BCA assay.

    I hope this information will be useful, but please let me know if you have further questions or if there is anything else that we can do for you, and I'll be happy to help.

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