Cell fractionation is a laboratory technique used to separate cellular components based on their size, density, or other physical properties. This process allows researchers to isolate and study specific cellular organelles or molecules. The steps involved in cell fractionation are as follows:
1. Cell Preparation:
The first step is to prepare the cells for fractionation. This involves growing or culturing the cells in appropriate media, maintaining optimal conditions for cell viability, and ensuring they are in a healthy state. The cells can be derived from tissues, cell lines, or organisms depending on the experimental requirements.
2. Cell Disruption:
Once the cells are prepared, the next step is to disrupt their membranes and release the cellular contents. This can be achieved through various methods, including mechanical disruption (such as homogenization or grinding), enzymatic digestion, or sonication. The goal is to break open the cells and release the intracellular components while minimizing damage to the cellular structures.
3. Homogenization:
Homogenization involves further breaking down the disrupted cells to obtain a homogeneous cellular suspension. Mechanical methods like high-speed blending or passing the cell suspension through a narrow gauge needle can be used. This helps to ensure a uniform sample and facilitate subsequent fractionation steps.
4. Differential Centrifugation:
The cell homogenate is subjected to a series of centrifugation steps to separate different cellular components based on their sedimentation rates. The initial centrifugation is performed at low speed to remove cell debris and unbroken cells. The resulting supernatant, known as the crude fraction, contains a mixture of organelles, membranes, and cytoplasmic components.
5. Density Gradient Centrifugation:
To further separate and purify specific organelles or components, density gradient centrifugation is employed. This technique utilizes a density gradient medium, such as sucrose or cesium chloride, layered in a centrifuge tube. The cell fraction is carefully layered on top of the gradient and subjected to high-speed centrifugation. During centrifugation, particles sediment through the gradient based on their density, resulting in distinct bands or fractions containing specific organelles or components.
6. Fraction Collection:
After centrifugation, the fractions containing the desired cellular components are collected carefully. This can be done by carefully aspirating or extracting the specific bands or layers from the gradient using a syringe or pipette. The collected fractions are then processed further for analysis, such as protein quantification, enzymatic assays, electron microscopy, or other biochemical techniques.
7. Analysis and Characterization:
The collected fractions are analyzed using various techniques to identify and characterize the isolated cellular components. This may involve protein analysis using gel electrophoresis, immunoblotting, or mass spectrometry, as well as microscopy, enzyme activity assays, or functional studies to determine the biological properties of the fractions.