Subcellular Fractionation Protocols Explained
The inside of all eukaryotic cells is divided into organelles and compartments, each with unique functions and unique protein populations. Subcellular fractionation allows you to separate these organelles and their protein populations from each other.
In this article, you will discover how subcellular fraction works and why you might want to use it in your research. You’ll also get a breakdown of how subcellular fractionation protocols work, links to commonly used protocols, and hear about different commercial subcellular fractionation kits.
Why You Need Subcellular Fractionation
If you are not yet using subcellular fractionation, you might be wondering how it can be used. There are various reasons for performing subcellular fractionation.
Learn About Your Favorite Protein’s Function
Function often correlates with where a protein can be found. Therefore, when characterizing a protein a good place to start is to identify where your protein resides using subcellular fractionation.
Increase Detection of Your Protein
Subcellular fractionation can help improve immunoprecipitation and Western Blot results. This is especially true if your protein is in low abundance or if your sample is complex, as cellular fractionation partially purifies your protein, increasing its concentration, which can improve your ability to detect it.
Remove Unwanted and Problematic Proteins
Cellular fractionation of complex mixtures can remove unwanted proteins that might interfere with your application. For example, it can remove proteins that compete for your antibody’s attention during immunoprecipitation.
How Does Subcellular Fractionation Work?
In general, different organelles and cellular compartments have different physical properties and knowing these properties is what makes cellular fractionation possible. Unique properties include size, shape and buoyant density. These unique properties are exploited in subcellular fractionation, which is most often accomplished by centrifuging organelles in a high viscosity media.
The details of your subcellular fractionation protocol matter, for one, not all protocols preserve protein activity.
Therefore, it is important that you choose the correct subcellular fractionation protocol for what you want to study: protein activity, organelle morphology, the organelle’s protein composition, etc.
But do not worry, whatever you want to study, there is a subcellular fractionation protocol out there.
Subcellular Fractionation Protocols Explained
While there are several subcellular fractionation protocols to choose from most share the following general steps.
Step 1: Lyse your cells
How you lyse your cells in subcellular fractionation is very important and depends on your protein type, the organelle or compartment you are interested in, and your downstream applications.
A hypotonic lysis protocol with low concentrations of non-ionic detergent is commonly used if you want to separate whole organelles, as hypotonic solutions will break the cell membrane (allowing you access to organelles), but leaves your cell’s nucleus and other compartments intact.
If, however, you are working with membrane proteins, cell lysis is best done with a detergent such as Triton X-114, maltoside or digitonin.
Both hypotonic and detergent lyses can also be combined with the use of dounce homogenizers, shakers or even sonication to aid in cell membrane disruption.
Step 2: Fractionate Your Lysed Cell’s Components
Now that your cells are lysed, you can separate the cellular components by their physical properties using centrifuging in a high viscosity media.
What kind of media you choose to use (e.g., sucrose, glycerol, or Percoll) depends on a number of factors, especially your downstream application.
There are always trade-offs when selecting a fractionation method, such as whether purity, protein activity, or yield is most important.
In general though, if you are doing assays that require enzymatic activity, time and temperature are your most important factors. Therefore if protein activity is your interest, you should avoid detergents (or use only a very low concentration) and you should use a faster protocol.
For example, if you want to purify mitochondria with functional mitochondrial proteins you might use sequential centrifugation: first using a sucrose medium at 5000 x g to collect the cytoplasmic fraction, followed by a second fractionation in Nycodenz gradient (17%, 25%, 35%, 50%) at >100,000 x g for a couple of hours to separate the peroxisomes from the mitochondria.
However, if you are interested in only protein composition, for example in proteomics, purity and quantity are your most critical factors.
To claim that a protein belongs to an organelle, you need as pure of a fraction as possible. Therefore, you should use density gradient centrifugation (e.g. Percoll), which requires a long (several hours to overnight) centrifugation but with high purity results.
Also, if you are primarily interested in purity as opposed to functionality, a small concentration of detergent can actually be beneficial, as detergents can facilitate the solubilization and separation of your cellular components.
Step 3: Collect Your Fraction of Interest
Fractions are collected by pipetting through the high viscosity media gradients with Pasteur pipettes or as pellets. Whatever the method, though, it is important that you are gentle. Always treat your pellet or fraction as if it was a bomb that will explode if treated casually.
This is the time to show off your best pipetting skills!
Step 4: Assess How Well You Did
Now that you think you have successfully separated and collected your cellular compartments into individual fractions, you need to verify your success.
If you have superman’s microscopic vision this would be a great time to use it! But if you are like most of us and don’t, fear not, there is another way. You can run your fractions on a Western blot and look for the presence of certain proteins markers to verify the purity of your fractions (Table 1).
Cytoplasm or cytoskeleton
Vimentin (non-epithelial cells)
Desmin (muscle cells)
Cytokeratin (all epithelial cells, some non-epithelial cells)
Golgin subfamily A member 2 orgalactosyl transferase
Cytochrome c oxidase 1
Lysosomal-associated membrane protein 1
Top Tips For Perfect Fractionation
Mind Your Time and Temperature
Be as quick as you can, and keep your buffers and samples at 4oC during all steps (unless otherwise stated in the protocol).
Don’t Forget Your Inhibitors
Remember to add both your protease inhibitors and your phosphatase inhibitors. Normally cells keep proteases and phosphatases restrained and tightly regulated but after cell lysis, all bets are off.
Freed proteases can cleave your proteins’ peptide bonds and destroy them. While phosphatases can remove the phosphates from phosphorylated proteins. This is bad because it may deactivate or alter your proteins’ interactions. So, always be sure to add fresh protease and phosphatase inhibitors to your samples and keep everything on ice!
Remember: Proteins Often Exist in Numerous Compartments
Tried and Tested Subcellular Fractionation Protocols
So you know you want to perform subcellular fractionation. You understand how it works. How do you find the perfect protocol for your needs? As mentioned above, fractionation protocols differ depending on your individual requirements so there is no one-size-fits-all approach. You need to find a protocol that fits with your sample type and your downstream applications.
So how do you find the right protocol? The literature is one great place to start. Below are links to some fractionation protocols we’ve uncovered, but there are many different ones available, so if none of the protocols listed here don’t fit your needs, just do some more research.
Protocols From the Literature
- Cox, B., Emili, A. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Nat Protoc. 2008 1, 1872–1878.
- Krapfenbauer K, Fountoulakis M, Lubec G. A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions. Electrophoresis. 2003;24(11):1847-70.
- Graham JM. Isolation of lysosomes from tissues and cells by differential and density gradient centrifugation. Curr Protoc Cell Biol. 2001;Chapter 3:Unit 3.6.
- Attardi G, Ching E. Biogenesis of mitochondrial proteins in HeLa cells. Methods Enzymol. 1979;56:66-79.
- Basrur V, Yang F, Kushimoto T, Higashimoto Y, Yasumoto K, Valencia J, et al. Proteomic analysis of early melanosomes: identification of novel melanosomal proteins. J Proteome Res. 2003;2(1):69-79.
- Welton JL, Khanna S, Giles PJ, Brennan P, Brewis IA, Staffurth J, et al. Proteomics analysis of bladder cancer exosomes. Mol Cell Proteomics. 2010;9(6):1324-38.
- Song Y, Hao Y, Sun A, Li T, Li W, Guo L, et al. Sample preparation project for the subcellular proteome of mouse liver. Proteomics. 2006;6(19):5269-77.
Subcellular Fractionation Kits
If you don’t have the time or resources to search the literature and test out protocols, there is an alternative. Many commercial life science companies offer subcellular fractionation kits. These ready-made kits can be a great way to get the results you need quickly.
If you are torn between using a commercial kit or finding a protocol from the literature we can help. Kits offer some great benefits but there are some costs to consider too.
The Pros of Commercial Fractionation Kits
Time is precious in the lab and your time also costs money. Using a premade kit can save you a lot of time in the lab as these kits are generally quick and simple to use, and have already been optimized. Just pick the right kit for your needs and away you go.
Kits have been rigorously tested and optimized during development and undergo quality testing to ensure each kit provides reliable and reproducible results. This gives you peace of mind that your results are comparable between days and kits. It also makes it simpler for other researchers to validate your results.
Using a kit makes it easier to write up your lab book and your paper. Instead of writing out a complicated protocol each time, you can just say ‘used X kit’ and detail any variations you made (if any) from the standard protocol. This also makes it easier when writing your paper.
The Cons of Commercial Fractionation Kits
As we’ve highlighted above, commercial kits for subcellular fractionation go through rigorous research and development phases and manufacturing is subject to rigorous quality controls. Companies need to make back these costs (along with profits) from the sale of the kit, meaning that kits are often significantly more expensive than DIY protocols from the literature.
Buffers in kits are generally premade and this makes it difficult to tweak or change them if needed. For example, buffers may contain ingredients, such as detergents, that are incompatible with your downstream applications. You may also find that while the kit works great on one sample type, it performs suboptimally for another, meaning you may need to have multiple kits for each sample time.
Kits are great, just follow the protocol, and away you go. But the downside is that you probably don’t understand what is happening at each step of the protocol, which can inhibit troubleshooting when things go wrong. While there is often a troubleshooting guide in the protocols, they might not cover every scenario.
Commercial kits are often full of single-use plastic which is discarded after use. These kits often also only process a small amount of sample, meaning if you need a large amount you may need to use multiple tubes, etc. per sample. Kits may also contain reagents you already have large stocks of in the lab, and would not have needed to purchase if using an alternative to kits.
How to Choose the Right Kit For Your Needs
Decided that a kit is the way you want to go? You can give yourself the greatest chance of success by considering the following points when selecting your kit.
Every sample type is different and this means that kits that have been optimized for one sample type may not work effectively on samples of a different nature. Check the kits descriptions or talk to a sales rep to check that the kit will work for your intended sample type. If you have multiple sample types, try to find a kit that has been tested or used (look in the literature) with all of them. Taking the time to do a bit of research now can save money and effort later.
Many kits have been created with a standard lab in mind and require only a benchtop centrifuge or other common equipment to use, but you should check that you have access to the necessary equipment before you purchase.
Kits may contain chemicals, such as detergents, that are incompatible with your needed downstream application. Check the manual or speak to a sales rep to check if the kit will work with your intended application.
Subcellular Fractionation Protocols Summarized
There is a wide range of protocols available for subcellular fractionation along with a selection of commercially available kits. Choosing between protocols depends on various factors including your sample type, budget, required sample volume, and downstream applications.
Whether you are purchasing a kit or searching the literature for a protocol, it is vital to carefully review the chosen method to ensure it fits with your experimental needs.
So, what do you think now? Are you ready to do subcellular fractionation?
Originally published May 2014. Reviewed and updated March 2022
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it was good & useful