Subcellular fractionation is the process separating the membrane-bound organelles within eukaryotic cells, which are found in the kingdoms Protista, Plantae, Fungi and Animalia. All eukaryotic cells share certain general features, including:
- They all have a nucleus.
- They are 10x larger than prokaryotic cells (which do not have an organized nucleus).
- They are enclosed by a plasma membrane.
- Their cytoplasm is made of cytosol and ribosomes.
- They have an internal cytoskeleton.
- Their extra-cellular matrix is comprised of proteins and glycoproteins.
- Locomotion is achieved through flagella or cilia.
Reasons for Subcellular Fractionation
There are a couple of key reasons why life science researchers need to conduct subcellular fractionation. The first is to learn more about a protein’s function and where it resides. The second is to improve the results of immunoprecipitations (such as removing unwanted proteins).
Protocol Selection
Prior to subcellular fractionation, researchers must determine what aspect of the organelles within eukaryotic cells they wish to study, such as protein activity, organelle morphology, protein composition, and so on. There are several protocols available to assist researchers once they have determined their research goals.
Subcellular Fractionation Protocol Steps
Generally, there are 4 subcellular fractionation protocol steps as follows:
- Step 1: Cell Lysis
The correct cell lysis method depends on a number of factors, including protein type, the organelle within the eukaryotic cells that researchers want to examine, and various downstream applications (e.g. protein purification, proteomics, X-ray crystallography, Western blotting, immunoprecipitation, etc.).
- Step 2: Subcellular Fractionation
Next, researchers use centrifuging in a high viscosity media (such as sucrose, glycerol or Percoll) in order to achieve subcellular fractionation. A number of factors must be taken into consideration here to ensure that the process is efficient, cost-effective, and yields the best possible results.
- Step 3: Collect Fractions
Researchers then collect fractions by a process of gently pipetting through the high viscosity media.
- Step 4: Assess Results
Lastly, researchers verify and assess their results by (for example) running fractions on a Western blot.
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