I do not have to tell you that eukaryotic cells are not just sacks of homogeneous protoplasm. Instead the inside of all eukaryotic cells are divided into organelles and compartments, each with unique functions and unique protein populations. And there may come a time when you need to separate these organelles and their protein populations from each other by subcellular fractionation. Read below to learn how and why you might perform subcellular fractionation.
Why You Need Subcellular Fractionation
You may need to do subcellular fractionations for numerous reasons: 1) If you want to learn about your favourite 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. 2) To improve the results of your immunoprecipitations, Western blot, etc. This is especially true if your protein is in low abundance or if your sample is complex. As performing cellular fractionation partially purifies your protein, which increases its concentration and can improve your detection of it. Additionally, cellular fractionation of complex mixtures can remove unwanted proteins that might interfere with your application — e.g. it can remove proteins that compete for your antibody’s attention during immunoprecipitation . 3) Because after successfully performing a cellular fractionation you will feel justified in imbibing in a cold beer or a glass of good wine.
Mile-High View of Subcellular Fractionation
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. All of which 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 matters, 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. (See the end of this article for some examples).
Getting Your Hands Dirty
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 are 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 interested in membrane proteins, cell lysis is best done with 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 – sucrose, glycerol, or Percoll – depends on a number of factors, especially your downstream application.
There are always trade-offs when selecting a fractionation method, purity vs protein activity vs yield. 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 not use detergents (or only a very low concentration of detergents) 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, e.g. in proteomics, purity and quantity are your most important 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 has better purity results. Also if you are primarily interested in purity opposed to functionality, 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 fractions as if it was a bomb which 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.
Compartments and their common protein markers:
- Cytoplasm or microtubule cytoskeleton: tubulin
- Golgi apparatus: golgin subfamily A member 2 orgalactosyl transferase
- Mitochondria: cytochrome c oxidase 1 or succinate dehydrogenase
- Chromatin: histones
- Endosomes: clathrin
- Lysosomes: Lysosomal-associated membrane protein 1
- 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 (both your protease inhibitors and your phosphatase inhibitors). Normally cells keep proteases and phosphatases restrained and tightly regulated but after cell lysis all beats 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!
- Proteins often exist in numerous compartments. It is very common for a protein to be in more than one compartment, but with distinct functions or interacting partners in each one. Some proteins even shuttle from one compartment to another, in processes related to the cell cycle stage or extracellular signals. So be very cautious, read the literature and plan your experiments accordingly. Also consider the fact that you may need to treat your cells with stimuli or drugs to localize your protein in the correct or consistent place. And if you find a protein localized to a place not known before, find out what it does over there and…publish it!
So, what do you think now? Are you ready to do subcellular fractionation?
Examples of subcellular fractionation protocols:
- Biogenesis of mitochondrial proteins in HeLa cells. PMID: 459888
- Isolation of lysosomes from tissues and cells by differential and density gradient centrifugation. PMID: 18228358
- Proteomic analysis of early melanosomes: identification of novel melanosomal proteins. PMID: 12643545
- Proteomics analysis of bladder cancer exosomes. PMID: 20224111
- Sample preparation project for the subcellular proteome of mouse liver. PMID: 16941572