Overview

  • Product name
    Cell Fractionation Kit - High Throughput (HT)
    See all Cell Fractionation kits
  • Assay type
    Direct
  • Assay time
    1h 00m
  • Species reactivity
    Reacts with: Other species, Mammals
  • Product overview

    Abcam's Cell Fractionation Kit - High Throughput (HT), provides a method and reagents for a simple and rapid preparation of cytosolic, mitochondrial and nuclear fractions from adherent cells.

    The Cell Fractionation Kit is designed to produce highly enriched fractions that allow for the monitoring of proteins of interest through various cellular compartments. It is not designed to produce purified cellular fractions.

    The kit is especially suitable for, but not limited to, high throughput fractionation of adherent cells in a 96-well plate format. The entire fractionation of 96 samples into the cytosolic, mitochondrial and nuclear fraction is achieved in one hour. The kit is based on sequential detergent extraction of cytosolic, mitochondrial and nuclear proteins without the need for mechanical disruption of cells, and thus fractionates adherent cells into cytosol-containing, mitochondria-containing and nuclei-containing fractions. These fractions are referred throughout the protocol as cytosolic, mitochondrial and nuclear fractions. The kit prepares sufficient sample material for subsequent Western blot analyses, dot blot analyses or for analyses by microplate ELISA assays.

     

    ab109718 is designed to allow the measurement of any proteins which are differentially represented in the cytosol, mitochondria and nuclei, and is particularly applicable to studies of proteins that translocate between these three cellular compartments.

     

    A second version of the kit is also available, ab109719 Cell Fractionation Kit - Standard. This version is designed for fractionating cells grown in batches and is ideal for experiments requiring larger sample amounts.

     

  • Notes

    Storage: Store Buffer A, Detergent HT II, Detergent HT III and 5X SDS Sample Buffer at -20°C. Store Detergent HT I at -80°C. Store the collection plates and seal films at room temperature.

Properties

Images

  • Measurement and distribution of proteins using Western blot, which are differentially represented in the cytosol, mitochondria and nuclei after treatment with varying concentrations of staurosporine.

Protocols

References

This product has been referenced in:
See 1 Publication for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

We have not tested localization of plasma membrane proteins or cytoplasmic organelles (besides mitochondria) with ab109718. However, we have tested distribution of these compartments using ab139409, a kit which works on the same principal as ab109718. Using ab109718 we found that plasma membrane, ER and golgi proteins are found mostly in the mitochondrial/membrane fraction. For blot images using ab139409 kit on selected targets, please see datasheets below:

https://www.abcam.com/ab139409.html
https://www.abcam.com/Cell-Fractionation-Kit-Standard-ab109719.html
https://www.abcam.com/Plasma-Membrane-Fraction-Western-Blot-Cocktail-ab139413.html
https://www.abcam.com/Organelle-Detection-Western-Blot-Cocktail-ab133989.html
https://www.abcam.com/membrane-fraction-wb-cocktail-ab140365.html
https://www.abcam.com/ab139415.html


If you plan to use ab109718 to fractionate cells into nuclear fraction vs everything else, I would recommend just to skip the extraction with the Detergent HT1.

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Answer

ab109718 is based on sequential and selective extraction of cytosolic, mitochondrial and nuclear proteins with detergents that allow sequential release of cytosolic and mitochondrial proteins to the extracellular buffer. In the first step, the plasma membrane is selectively permeabilized with Detergent I. The cytosol-containing fraction is separated from the remainder of cells containing intact mitochondria and nuclei by a simple centrifugation step. In the second step, mitochondrial proteins are then extracted with Detergent II and separated from the nuclei containing fraction by a second centrifugation step. The exact steps can be found in the following parts of the protocol:

Cytosol fraction
pg 9
Step 6.6-6.8

Mitochondria fraction
pg 10
Step 6.9-6.11

Nuclear fraction
pg 10
Step 6.12-6.13

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Answer

Thank you for contacting us and you interest in our products.
Unfortunately I can now confirm that the Cell Fractionation Kit - HT (ab109718) is not suitable for use with tissue samples. However, we do have some other kits which you may want to consider:
The Mitochondria Isolation Kit for Tissue (ab110168 and with dounce homogenizer ab110169) can be used to isolate the mitochondrial fraction from tissue samples.
The EpiSeeker Nuclear Extraction Kit (ab113474 and Nucleic acid free, ab113477) enables the isolation of nuclear proteins from cell or tissue samples.
I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

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Answer

Thank you for your enquiry and your interest in our products.

I can confirm that both kits can be used for mammalian cells (including mouse samples).

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you fro your enquiry.

I am sorry to confirm we would not recommend to use ab109718 Cell Fractionation Kit - HTor ab109719 Cell Fractionation Kit Standardfor isolation of mitochondria from tissue. They are designed for cell culture single cell suspension samples only. The issue with solid tissue is in the difficulty to prepare the starting material for the fractionation: single cell suspension without breaking the cells.

If you want to isolate mitochondria, then I can recommend to consider ab110168 or ab110169 instead. These are designed for mitochondrial isolation from tissue.

https://www.abcam.com/index.html?datasheet=110168 (or use the following: https://www.abcam.com/index.html?datasheet=110168).

https://www.abcam.com/index.html?datasheet=110169 (or use the following: https://www.abcam.com/index.html?datasheet=110169).

The important steps to consider from the protocol provided on the datasheetare as follows:

Spin 1 isolates the unbroken cells and nucleiSpin 2 isolates the mitochondria. The remaining supernatant after spin 2 contains mostly cytosolic proteins –the protocol suggests to save this supernatant sample (step 12) and these could be used.

There are wash steps to clean up the mitochondria (Steps 13-15) However the supernatant sample could also be washed by spinning at 12000g repeatedly and collecting the supernatant each time.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

Thank you for calling Abcam earlier. For the Mitochondria Isolation Kit for Cultured Cells (ab110170), if you want to isolate the cytosolic fraction, you could keep the supernatant after 'SPIN 2' (see attached protocol) and that would give you a crude fraction. However there is no guarantee that this will give you clean preps without damaging the RNA. I would however recommend ab109718, the Cell Fractionation Kit as this will give you cleaner fractions of the mitochondria and cytosol. Also in the protocol for that product, it gives you the volumes to use for a 24 well plate and you get enough reagents for 1x96 well plate. Please let me know if there is anything else I can help you with.

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Answer

This is quite a common question. The fractionation kit is good for easily and rapidly separating cytosol, Mitochondria and nuclei.  This is a great method for making fractions to follow movement of proteins between these compartments such, but not limited to, in apoptosis. To purify Mitochondria and subsequently purify its compartments I would recommend starting with our mitochondria isolation kit ab110169. I hope this helps. Please contact me again if you have any further questions.

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Answer

I now have some further information relating to the Cell Fractionation Kit - HT (ab109718) we discussed over the phone. The fractionation kit is good for easily and rapidly separating cytosol, Mitochondria and nuclei.  This is a great method for making fractions to follow movement of proteins between these compartments such as, but not limited to, in apoptosis. However, these fractions are only separated from each other and none of these fractions should be considered pure of other cellular components. Therefore to purify Mitochondria and subsequently  purify its compartments I would recommend starting with Mitosciences Mitochondria isolation kit for Cultured Cells ab110170 (which also comes with Dounce Homogenizer ab110171 ). If you want to isolate Mitochondria from tissues ab110168 would be more appropriate (with Dounce Homogenizer ab110169). These kits isolate the Mitochondira through centrifugation and resuspension in a buffer. This isolated suspension should be compatible with further isolation of the Mitochondrial compartments. I hope this information has been of help to you. If you have any further questions please do not hesitate to contact us again.

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Answer

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products. We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase. This kit has been used in the protocol from a poster presented at the combined Keystone Symposia Cell Death Pathways and Mitochondrial Dynamics and Physiology, March 22-27, 2009 (see attached).  You can see how it was used for this. I hope this information is helpful. Please do not hesitate to contact us again with any other questions.  

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Answer

Thanks so much for contacting us and for your interest in ab109718. We only have the full size kits for sale and don't have sample sizes, however we fully guarantee this kit to work as stated on the datasheet for up to 6 months after purchase. If it did not work as stated, we would offer a replacement or refund. I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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