Differential And Density Gradient Centrifugation

What is Differential Centrifugation ?

Differential centrifugation is a very common procedure in biochemistry and cell biology. Used to separate organelles and other subcellular particles based on their sedimentation rates. Although frequently used in biological analysis, differential centrifugation is a versatile technique. Also suitable for crude purification of non-living suspended particles (e.g. nanoparticles, colloidal particles, viruses). In a typical case of differential centrifugation used to analyze cellular biological phenomena such as organelle distribution, tissue samples are first lysed to disrupt cell membranes and release organelles and cytosol. The lysates were then subjected to repeated centrifugation. Where particles settle quickly enough for a given time at a given centrifugal force form a dense “particle” at the bottom of the centrifuge tube.

Cell Fractionation By Differential Centrifugation

For a typical cell homogenate, 10 minutes. Spinning at low speed (400-500 x g) yields pellets consisting of intact tissue, whole cells, nuclei, and large fragments. Low velocity projectiles are traditionally called nuclear projectiles. A 10 minutes. Rotate at moderate speed, generating a force of 10,000 to 20,000 x g to bring the mitochondria down along with the lysosomes and peroxisomes. Therefore, the second particle in the traditional cell fractionation scheme is called the mitochondrial particle.

Further separation of cells by differential centrifugation requires the use of an ultracentrifuge. This instrument is designed to rotate the rotor at high angular velocities to generate very high g-forces. Air must be pumped out of the chamber to avoid heat generation due to air friction. In fact, many rotors designed for ultracentrifuges aren’t even aerodynamically designed because they spin in a vacuum. A one-hour high-speed ultracentrifuge run generating approximately 80,000 x g force produces microsomal particles. Microsomes include membrane fragments, including cell membranes and endoplasmic reticulum. When ruptured in aqueous media, membrane fragments form vesicles, so inspection reveals many membrane vesicles of varying sizes. Due to the different protein content, the vesicles themselves can be separated according to density. But that’s the subject of another file.

Spinning at around 150,000 xg for hours, you can break down even the largest macromolecules. The remaining supernatant consists of soluble components of the cytoplasm, including salts, small and precursor molecules, and dissolved gases.

Process Of Differential Centrifugation

The centripetal force created by centrifugation can be hundreds or thousands of times greater than gravity, greatly speeding up the process. The higher the revolutions per minute (RPM), the higher the gravity. If all we can do is drive suspended particles to the bottom of the tube, then the usefulness of centrifugation in cell separation will be limited. However, because of the physics of suspended particles, the researchers were able to control the size of the downed particles.

In a suspension of circular particles of equal density but different diameters, the force driving a given particle to the bottom is equal to its mass times the acceleration applied. The volume of a particle is a function of its radius, and its mass is equal to its volume multiplied by its density coefficient, which is a constant. The volume of a sphere is equal to 4/3 times pi (a constant) times the radius cubed. For a suspension of spherical particles of equal density under a specific set of conditions, the only variable that determines the force on a given particle is its radius.

The resistance to motion through the solution is proportional to that portion of the surface area that is pushed through the medium. For particles of similar shape, smaller particles encounter less resistance than larger particles. Since the surface area of ​​a sphere is 4 times pi times the radius squared, and 4 times pi is a constant, then for spherical particles of the same composition, the only variable that determines resistance under given conditions is the particle.

The driving force increases proportionally to the cube of the radius. Movement resistance increases proportionally to the square of the radius. It is not difficult to see that as the radius of the particle increases, so does its tendency to approach the bottom. Add in a lot of “resistance,” and the gravity experiments attributed to Galileo didn’t work well after all. Because large particles settle faster than small particles, researchers can separate large particles from small organelles, cells, etc. by simply controlling how long and how fast the centrifuge runs.

Describe Centrifugation Conditions

Few more information than centrifugal force, time, and temperature need to be reported when describing the materials and methods of centrifugation runs. The required speed (rpm) depends on the centrifuge and rotor used, which will vary from laboratory to laboratory. Therefore, reporting the centrifuge brand, rotor type, or speed is rarely relevant.

What Is Density Gradient Centrifugation?

The centrifugation process allows scientists to separate substances based on their shape and size. The sample is put into a centrifuge—a machine designed to spin liquid solutions at high speeds. Mixing or spinning subjects the mixture to centrifugal force, pushing larger particles from the center toward the bottom and smaller particles toward the top. Larger components are more responsive to forces than those smaller components.

In density gradient centrifugation, the process is similar. The samples are still put into the centrifuge, but the ultimate goal is not to sort by size. The spin of the centrifuge causes the denser particles to move to the outer edge. These particles have more mass and are carried farther due to their inertia. The less dense particles then settle towards the center of the sample. This creates a sorted solution layered by particle density from smallest to largest.

The Principle Of Density Gradient Centrifugation

Each particle has a specific set of physical properties; properties of its biological composition that can be used for separation and isolation. Density gradient centrifugation focuses on two – size and density. The length of time required for this process depends on the size of the particles. Larger particles reach their stable positions earlier, while smaller particles take longer to pass through larger particle regions and occupy positions deeper in the gradient.

Density Gradient Reagents

A reagent is any mixture or substance used in chemical analysis or experiments. In density gradient centrifugation, reagents are products used to help separate or separate cells. These products not only speed up the process, but also improve purity and yield. These reagents can greatly increase the efficiency of density gradient centrifugation by preventing particle aggregation, creating a fixed divider, or eliminating residual red blood cells.

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