Proteomics/Protein Separations - Centrifugation/Differential Centrifugation
Differential Centrifugation is a process involving multiple centrifugation steps, increasing the speed of centrifugation each time, used to separate cellular materials. This process uses a equilibrium density gradient. The mixture of cellular materials (known as homogenate) is separated into different layers based on the molecular weight, size, and shape of each type of molecules. Subsequent rounds of centrifugation will pellet first the nuclei, followed by the mitochondria, then the lysozomes, and finally the microsomes. The soluble fraction of the homogenate is what remains after these materials have been removed. To begin proteomics research focused on proteins found in one of the various differential fractions, that particular layer would first be isolated and collected and then further purification/experimentation can be carried out. A protocol for membrane protein extraction using multiple rounds of centrifugation at varying speeds can be found at http://www.westernblotting.org/protocol%20membrane%20extraction.htm .
Each fraction obtained is a partially-pure preparation from which contaminating particles can be removed by repeated washing. This is accomplished by first resuspending the pellet in an isotonic solvent and then re-pelleting using centrifugation. To purify the sample further is may then be separated using density gradient centrifugation.
The nuclear fraction of the homogenate can be pelleted by centrifugation at 600g for 10 minutes. This fraction will contain all material found in the nucleus including genomic DNA, proteins involved in replication and transcription, as well as other nuclear proteins and materials.
The mitochondrial fraction of the homogenate is obtained in the pellet when the post-nuclear supernatent (i.e. the supernatent of the first centrifugation ) is centrifuged at 5000g for 10 minutes. The mitochondria are sometimes called the “power house” of the cell because of their role in energy production. Mitcohondria also have their own DNA unique from that which is found in the nucleus. Proteins of interest found in the mitochondria would be those involved in the conversion of organic materials into ATP through oxidative phosphorylation.
The lysosomal fraction can be pelleted by centrifugation at 12,000xg for 5 minutes. Lysozomes contain important proteins for breaking down cellular waste.
The microsomal fraction is the pellet produced when the postmitochondrial supernatent is centrifuged at 50,000g for 60 minutes. Microsomes are small vesicles enclosed by a biological membrane. During centrifugation the endoplasmic reticulum is fragmented into microsomes.
The soluble fraction of the homogenate is the supernatant that remains after the other materials have been pelleted out in previous rounds of centrifugation. Within this fraction soluble proteins are found. To further isolate/purify proteins found here a different method of centrifugation can be used, such as density gradient centrifugation. In fact, density gradient centrifugation can be used to further separate any of the fractions collected by differential centrifugation.
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