by Caroline Aufgebauer
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Difficulty Rating: 3/5
What is the general purpose? Cellular fractionation is a technique used to separate individual compartments of cells. These cellular compartments can be thought of as individual rooms of a factory. Each room is specially equipped for a specific function, such as a warehouse stocked with shelving units and heavy lifting machinery to store products. By separating individual cellular compartments, scientists can better understand their unique functions and what kinds of specialized tools they contain.
Why do we use it? Cells are made up of multiple individual compartments each containing different kinds of proteins (Figure 1). The three main subcellular compartments that scientists study are the cytoplasm, nucleus, and membrane-bound organelles. The cytoplasm is the semifluid substance that fills the inside of a cell, and it is made up of water, salts, and proteins. The nucleus stores the cell’s genetic information and is enclosed by a membrane that separates it from the cytoplasm. Membrane-bound organelles are specialized parts within the cell that perform a specific function, much like the organs in our body. Examples of membrane-bound organelles are the mitochondria, which produces energy for the cell, and lysosomes, which break down cellular waste.
These individual cellular compartments are made up of unique protein populations that determine their function. For example, the nucleus contains proteins that copy the genetic information from DNA into RNA messages, while the mitochondria contains proteins that work together to produce energy in the form of ATP. When cells are stressed, they often adapt by moving proteins to new locations within the cell. Thus, cellular fractionation allows scientists to not only study protein populations in specific cellular compartments, but also to study how these populations change in response to stress.
Figure 1: Cells are made up of multiple cellular organelles that each perform a specific function. Image created by author using BioRender.
How does it work?
Overview
A cellular fractionation protocol isolates individual compartments, or “fractions,” of the cell step-by-step (Figure 1). These fractions are the cytoplasmic fraction, followed by the organelle fraction, and finally the nuclear fraction. A western blot is then used to identify what kinds of proteins are contained in these individual fractions.
Steps
1. Lysis of Cell Membrane
In order to access the inner components of a cell, we first break down the cell membrane in a process called lysis. We lyse the cells using a detergent called digitonin. The resulting mixture is referred to as cell lysate. The type of detergent is very important in every step. Digitonin is a detergent that breaks down the outer cellular membrane without affecting the membranes of cellular organelles, such as the nuclear membrane. This allows us to break open the cell and access the cytoplasm without breaking open any organelles.
2. Centrifugation (Isolation of the Cytoplasmic Fraction)
At this point, the lysate contains a mixture of cytoplasm and intact membrane-bound organelles and nuclei. Next, we separate the cytoplasm from the organelles and nuclei. To do this, we centrifuge the cell lysate. A centrifuge spins the lysate really fast and separates the components of the lysate based on density. In the lysate, the nuclei and membrane-bound organelles are much more dense than the cytoplasm. Thus, these organelles settle at the bottom of the tube and form a pellet, while the cytoplasmic fraction remains in solution. This allows us to specifically isolate the cytoplasmic fraction.
3. Lysis of Membrane-bound Organelles
Next, we isolate the components of membrane-bound organelles for our organelle fraction. To do this, we lyse the organelles using a different type of detergent called igepal. This detergent breaks down the membrane of organelles like the mitochondria and endoplasmic reticulum, but not the nucleus. This allows us to isolate the inner components of these membrane-bound organelles while keeping the nucleus intact.
4. Centrifugation (Isolation of the Organelle Fraction)
At this point, the lysate contains a mixture of the inner components of membrane-bound organelles and intact nuclei. Next, we separate the components of the membrane-bound organelles from the nuclei. To do this, we once again centrifuge the lysate. Now that we have broken apart the membrane-bound organelles, they are much less dense than the intact nuclei. Thus, centrifugation of the lysate will cause the intact nuclei to settle and form a pellet at the bottom of the tube, while the organelle fraction will remain in solution. This allows us to specifically isolate the organelle fraction.
5. Lysis of Nuclei
Next, we want to isolate the inner components of nuclei. To do this, we lyse the nuclei using two different detergents called sodium deoxycholate and sodium dodecyl sulphate. Together, these detergents effectively break down the nuclear membrane and release its inner components.
6. Centrifugation (Isolation of the Nuclear Fraction)
At this point, the lysate contains a mixture of the inner components of nuclei as well as insoluble proteins. Next, we separate the components of the nuclei from any insoluble proteins. To do this, we centrifuge the lysate, causing insoluble proteins to settle and pellet at the bottom of the tube. We can now easily isolate the nuclear fraction.
7. Western Blot
Finally, we want to identify which cellular fraction our protein of interest is located in. To do this, we perform a western blot. A western blot is a technique that allows scientists to detect the presence and relative amount of protein within a sample. In this case, a western blot can be used to detect the amount of our protein of interest in the cytoplasm, nucleus, and membrane-bound organelles of our cells.
Figure 2: Experimental workflow for fractionation of cells. Individual fractions are isolated through a series of lysis and centrifugation steps. Image created by author using BioRender.
