Overview

  • Product name
    Cell Fractionation Kit - Standard
    See all Cell Fractionation kits
  • Assay type
    Direct
  • Product overview

    Abcam's Cell Fractionation Kit (Standard) allows for the rapid and simple preparation of mitochondrial, cytoplasmic and nuclear containing fractions from cultured cells from mammals and other species.


    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.


    This kit does not require mechanical disruption of the sample. This is important as mechanical disruption can disrupt mitochondrial membranes, causing the biologically-irrelevant release of proteins. This kit is particularly useful in studying apoptosis and the movement of pro-apoptotic proteins such as cytochrome c.


    Sufficient materials are provided for fractionation of 1 x108 cells or for preparation of 40 samples, each corresponding to one 100 mm plate at 2.5 x 106 cells/plate.


    A companion product is also available which allows for the fractionation of cells grown in 96-well plates: Cell Fractionation Kit HT (ab109718).

  • Notes

     

Properties

Images

  • Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (5) nuclear fraction lysate. Membrane was blotted with a Plasma Membrane Fractionation WB cocktail ab140365 containing anti-Sodium Potassium ATPase antibody (110 kDa), anti-GAPDH antibody (37 kDa) and anti-Histone H3 (di methyl K9) antibody.

  • Cytosolic (C), mitochondrial (M) and nuclear (N) fractions of HepG2 cells were prepared as described in the Protocol. Fractions were analyzed by Western blotting using Abcam's ApoTrack™ Cytochrome c Apoptosis WB Antibody Cocktail (ab110415) containing antibodies against mitochondrial matrix (pyruvate dehydrogenase subunit E1α, PDH E1α), mitochondrial inner membrane (F1-ATPase α), mitochondrial intermembrane space (cytochrome c) and cytosolic (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) markers as well as with antibodies against additional mitochondrial matrix (Hsp70) and nuclear (poly (ADP-ribose) polymerase, PARP and SP1) markers, followed by appropriate HRP-conjugated goat secondary antibodies and ECL detection.

  • In this experiment, apoptosis was induced in Jurkat, HeLa and 143B osteosarcoma cells by treatment with staurosporine or in Jurkat cells by FAS. Mitochondrial and cytoplasmic fractions were isolated (using ab109719) and probed using ab110415. As is clear from the gels, cytochrome c has translocated partially in FAS-induced cells and STS-treated osteosarcoma cells, and almost completely in STS-treated Jurkat and HeLa cells. The three control targets allow for verification of the "cleanness" of the cell fractionation.

Protocols

References

This product has been referenced in:
  • Lee MH  et al. Inhibitory Effects of Menadione on Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation via NF-?B Inhibition. Int J Mol Sci 20:N/A (2019). Read more (PubMed: 30866458) »
  • Chen LY  et al. Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function. Osteoarthritis Cartilage 26:1539-1550 (2018). Read more (PubMed: 30031925) »
See all 33 Publications for this product

Customer reviews and Q&As

1-10 of 40 Abreviews or Q&A

Excellent for nuclear extraction

Excellent Excellent 5/5 (Ease of Use)
Abreviews
The kit was used to extract nuclear fraction from whole cell lysates. The mitochondrial fraction was not used. Lamin B1 was used as a nuclear marker and a strong signal was observed in the whole cell extract (WCE) and nuclear fractions (NUC) but not the cytoplasmic fraction (CYTO). Nuclear DNA was only sheared by vortexing and therefore the DNA present in the nuclear sample did cause smearing of the bands. Sonication could improve this but this was enough to demonstrate that the kit could quickly and efficiently purify nuclear fractions.

Abcam user community

Verified customer

Submitted Mar 31 2017

Got clean nuclear fraction

Good Excellent 5/5 (Ease of Use)
Abreviews
The kit is quite easy to use. I got a clean nuclear fraction (N) with positive CREB staining and negative tubulin staining. However, the cytosolic fraction (C) has nuclear contamination (CREB positive).

Ms. Simin Li

Verified customer

Submitted Dec 17 2016

Cell fractionation with Hela cells

Excellent Good 4/5 (Ease of Use)
Abreviews
Follow the protocol in general, but wash with Buffer 1 between different extractions.

Abcam user community

Verified customer

Submitted Jan 14 2016

Answer



I an confirm that the ab109719 is a cell fractionation kit and it has the word standard in its name to distinguish this product from ab109718 (Cell Fractionation Kit –HT), where the HT stands for high throughput. I believe that we did not have inquiry of this type so far, but I will bring this to discussion.


The recommended amount of cells to fractionate is 2.5X10E6 (in the protocol as discussed on the telephone).

The fractions produced by this kit are compatible with IP. The cytosolic and the mitochondrial fractions contain already detergent-solubilized proteins. The nuclear fraction is a nuclear suspension and thus needs to be extracted with a detergent of choice (not provided) to solubilize the nuclear proteins.

For IP, it is also possible to extract all three final fraction with detergent of choice. The amount of cells used for the fractionation can be scaled up or down according to user requirements, it is important however to keep the concentrations constant. If you plan to do IP with the fractions you will needs to decide on the amount of samples needed for the IP. There is virtually no loss of unaccounted biological material and the fractions are proportional to the original cell suspension. For example, as in protocol, cell suspension at 3.3x10E6 cells/ml will yield cytosolic fraction of concentration corresponding to 3.3x10E6 cells/mL.

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Answer

ab109719 is not suitable for this purpose, this kit is based on detergent fractionation of cellular proteins into three fractions. RNA distribution was not tested with this kit.



Mitochondrial isolation kits (ab110168 – ab110171) are designed to isolate whole mitochondrial organelle, as such these kits are suitable to isolate mRNA that is attached to/ within this compartment. I would recommend to supplement all the buffers of the Mitochondrial Isolation Kit with RNAse inhibitors. We do not have any data regarding the yield of mRNA. The reference below maybe useful reading:



http://www.ncbi.nlm.nih.gov/pubmed/6197615

Attardi G, Montoya J.

Methods Enzymol. 1983;97:435-69. No abstract available.

PMID:

6197615



Please let me know if you have any further questions!

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Answer

The kit we recommend for preparing subcellular fractions is ab109719 (or ab109718 for cells in 96-well plates).

Click here (or use the following: https://www.abcam.com/index.html?datasheet=109719).

The procedure uses only detergent, without any homogenization or sonication. It begins by lysing the plasma membranes, releasing the cytosol, mitochondria, and nuclei. Then the mitochondria and nuclei are spun down in a centrifuge, and the mitochondria are lysed, leaving the nuclei intact.

Another procedure is described at the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=11473

The instructions are a little confusing but basically, you are lysing the plasma membranes of the cells with a hypotonic buffer that does not lyse the nuclear membranes. The nuclei are spun down in a centrifuge, the supernatant (cytosol and small organelles like mitochondria) is removed, and then the nuclei are lysed by adding buffer that contains SDS at a final concentration of 0.1% and glycerol at a final concentration of 10%.

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Answer

The product is guaranteed to work with the cell concentrations as set in the protocol. However, it is certainly possible to create fractions of higher protein concentrations. This may need however some optimization. I would suggest to customer to try the kit as in the protocol first and only if the target concentration is below the detection, then try to optimize the procedure. The optimum extraction of proteins depends on detergent to total protein ratio. If the customer wants to receive fractions twice as concentrated, I would use not only twice as concentrated cells but also double the concentrations of Detergent I and II. These settings however may not work optimally. So it would be best to titrate the concentration of Detergent I in a first experiment and analyze the C and M fractions with cytosolic and mitochondrial markers. In the second experiment, take the best concentration of the Detergent I, titrate the Detergent II and analyze the M and N fractions with mitochondrial and nuclear markers. Similar optimization procedure is described in ab109718 (https://www.abcam.com/cell-fractionation-kit-ht-ab109718.html)

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Answer

Thank you for contacting us and your interest in our products. I am sorry for the delay in getting back to you.
I have consulted with the lab over the suitability of the two isolation kits, ab109719 and ab110170, in regards to your customers problem with plasma membrane contamination. They have confirmed the following:
When ab109719 is used, the plasma membrane and mitochondria co-fractionate together, so ab109719 would definitely not recommended to isolate plasma membrane-free mitochondria.
I would recommend to use ab110170 as a first step to isolate the mitochondria. However the mitochondrial fraction prepared using this kit will still heavily contaminated with the plasma membrane. As an example, see the attached Western blot analysis of fractions isolated from mouse heart homogenate (MHH), including isolated mitochondria and supernatant 2, using a similar method (ab110168, a tissue version of ab110170) with plasma membrane marker (Sodium Potasium ATPase).
In order to isolate plasma membrane-free mitochondria, gradient centrifugation (for example percoll/metrizamide, sucrose, ficol) must be used on the crude mitochondria fraction. There are variety of protocols to achieve this, my favorite: Methods in Enzymology, vol 182, 203-225 (1990). If you are concerned with plasma membrane contamination and want to follow mitochondria as well as plasma membrane marker, I would recommend to use the ab133989 western blot cocktail.
I hope this has been helpful. Please do not hesitate to let me know if you have any further questions.

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Answer



The appropriate fractionation depends on the ratio of detergents to the cellular mass. Since cells vary in their size, the extraction condition may need a optimization for a particular cell line. The customer reports good separation of cytosolic fraction, so I would recommend to keep the protocol steps 6.1 – 6.12 without a change. Since the customer reports that a nuclear marker protein is found mainly in the mitochondrial fraction, I would recommend to optimize the separation of mitochondrial and nuclear fractions by lowering the concentration of Detergent II to extract mitochondria. This can be done by preparing multiple tubes (aliquots) of cytoplasm depleted cells (steps 6.1-6.12). At that time, have ready multiple aliquots of Buffer C with variable concentration of Detergent II (for example Detergent II diluted 25x, 37.5x, 50x, 75x, 100x in buffer A). Then finish the protocol (steps 6.14 and up) with parallel multiple fractionations obtaining M and N fractions for each Detergent II extraction conditions. Analyze multiple M and N fractions by WB using nuclear and mitochondrial markers and select the condition that have best separation of mitochondrial and nuclear markers in M and N fractions.

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Answer

This product was updated in September 2012 to change the Buffer A from 2 bottles of 1X, to 1 bottle of 2X to streamline the packaging. I will inquire with product updates to see why the protocol was not changed. The 2X is simply diluted 1:1 with H2O to 1X. I have attached the updated protocol for you. I should mention also that the 2X buffer needs to be equilibrated to room temp prior to diluting to 1X with H2O. No other changes were made to the product or the protocol.

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1-10 of 40 Abreviews or Q&A

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