Cell Fractionation Kit - Standard (ab109719)

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.

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!

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/Cell-Fractionation-Kit-Standard-ab109719.html).

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%.

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)

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.

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.