CsCl (Cesium chloride) is used in density gradient centrifugation primarily because it can form a stable and self-generating density gradient under centrifugal force, allowing for the separation of biomolecules like DNA, RNA, and proteins based on their buoyant densities.
Detailed Explanation
Here's a more detailed breakdown of why CsCl is so effective:
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Formation of a Density Gradient: When centrifuged at high speeds, CsCl forms a continuous density gradient. The Cs+ ions migrate under the centrifugal force, creating a region of higher concentration (and therefore higher density) towards the bottom of the tube and a region of lower concentration (and lower density) towards the top.
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Density Near DNA: CsCl can achieve densities very close to that of DNA. This is crucial for high-resolution separation of DNA molecules with subtle density differences.
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Separation Based on Buoyant Density: During centrifugation, biomolecules like DNA, RNA, and proteins will migrate through the CsCl gradient until they reach a point where the density of the surrounding CsCl solution equals their own buoyant density. At this point, they will band or form a distinct layer.
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Separation of DNA, RNA, and Proteins: Because DNA, RNA, and proteins have different buoyant densities, they will band at different locations within the CsCl gradient. This allows for their separation. Generally:
- Proteins: Typically sediment in the heaviest, bottom-most layer of the gradient due to their higher density.
- DNA: Bands at an intermediate density range.
- RNA: Being lighter, tends to be present at the topmost layer with the lowest density.
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Self-Generating Gradient: The beauty of using CsCl is that the gradient forms itself during centrifugation. This eliminates the need to pre-form gradients, simplifying the process.
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High Resolution: CsCl gradients offer very high resolution, making them suitable for separating molecules with slight differences in density, such as DNA fragments with different GC content or DNA molecules with different isotopes.
In summary, CsCl's ability to form a stable, self-generating density gradient close to the density of biomolecules, coupled with its high resolution, makes it ideal for separating DNA, RNA, and proteins in density gradient centrifugation. It separates molecules based on their buoyant densities in the gradient.
