Structural Biochemistry/Proteins/Purification/Differential Centrifugation
Purpose:you have the protein in some cells. Then, you want to remove the other protein to get the one you one.
Differential centrifugation is a method used to separate the different components of a cell on the basis of mass. The cell membrane is first ruptured to release the cell’s components by using a homogenizer. The resulting mixture is referred to as the homogenate. The homogenate is centrifuged to obtain a pellet containing the most dense organelles. Compounds that are the most dense will form a pellet at lower centrifuge speeds while the less dense compounds will likely remain in the liquid supernatant above the pellet. Each time, the supernatant may be centrifuged at faster speeds to obtain the less dense organelles. Performing centrifugation in a stepwise fashion, in which the centrifugation speed is increased each time, allows the components to be separated by mass. The rather dense nucleus is most likely to be found after the first centrifugation step, followed by the mitochondria, then smaller organelles, and finally the cytoplasm, which may contain soluble proteins.
Equilibrium sedimentation uses a gradient of a solution to separate particles based on their individual densities (mass/volume). A pivotal aspect about this type of sedimentation is that it is completely independent of the shape of the molecule. It is used to purify the differential centrifugation. A solution is prepared with the densest portion of the gradient at the bottom. Particles to be separated are then added to the gradient and centrifuged. Each particle proceeds until it reaches an environment of comparable density. Such a density gradient may be continuous or prepared in an incremental fashion. For instance, when using sucrose to prepare density gradients, one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose and add further less dense layers above. The homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken cells, is then layered on top. After centrifugation typically for an hour at about 100,000 x g, disks of cellular components residing due to the change in density can be observed from one layer to the next. By carefully adjusting the layer densities to match the cell type, specific cellular components can be enriched.
Sedimentation equilibrium is quite useful because a pellet is not formed. The speed of rotation creates enough force to make the protein leave the rotor, but it doesn’t condense it into a pellet. This is because a gradient in the concentration of the protein is produced. Diffusion reacts to counter the creation of the gradient and after a certain amount of time, a perfect balance between sedimentation and diffusion is achieved.
Sedimentation equilibrium is also practical to study the interactions between proteins. In particular it is used to ascertain the native state or native conformation of the protein. The native state tells us the exact structure in three dimensions. This information includes if it is a monomer, dimer, trimer, tetramer, etc. A monomer is a protein made up of one subunit. A dimer is two protein subunits that are rotated 180 degrees. A trimer is three subunits etc. This type of experimentation also allows us to determine whether the proteins can form oligomers (identical polypeptide chains tha make up two or more units of a protein). Additionally, the use of sedimentation equilibrium is that it determines equilibrium constants for protein-protein and protein-ligand interactions. The value of this Kd is often between 1nM-1mM. This is calculated by measuring the equilibrium constant (Kd). A final use of this is to determine stoichiometric ratios between protein complexes. An example of this is a ligand and its receptor or an antigen-antibody pair
- Berg, Jeremy (2006). Biochemistry (6th Ed. ed.). W. H. Freeman. ISBN 0716787245.
Durdik, Jeaninne. "Sedimentaion Equilibrium". Alliance Protein Laboratories. http://www.ap-lab.com/sedimentation_equilibrium.htm. Retrieved 2009-10-10.