Overview

  • Product name
    Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer)
    See all Mitochondria Isolation kits
  • Assay type
    Quantitative
  • Species reactivity
    Reacts with: Other species, Mammals
  • 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

  • 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.

Protocols

References

This product has been referenced in:
  • Paredes F  et al. Poldip2 is an oxygen-sensitive protein that controls PDH and aKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer. Proc Natl Acad Sci U S A 115:1789-1794 (2018). Read more (PubMed: 29434038) »
  • Uchihara Y  et al. Taxodione induces apoptosis in BCR-ABL-positive cells through ROS generation. Biochem Pharmacol 154:357-372 (2018). Read more (PubMed: 29859988) »
See all 13 Publications for this product

Customer reviews and Q&As

1-10 of 25 Abreviews or Q&A

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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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
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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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
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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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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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
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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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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Answer

If you are planning to use our activity kits to measure complex I activity,there is no needto keep the mitochondria viable. Whole mito preparations may be frozen at -80C and thawed at a later stage for enzyme activity. Howeverfor respiration experiments, the mitochondria must be tested within a couple of hours of isolation. Beyond that, membrane potential and respiration may be lost or it may decrease significantly.

I hope this is helpful. Please contact me again if you have any further questions.

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1-10 of 25 Abreviews or Q&A

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