Can form hydrophobic bonds with hydrophobic regions of proteins. Temperature requirements for cell fractionation Performed at 4 C to reduce the activity of proteases. All media and apparatus should be precooled and maintained at 4 C. Unwanted organelle damage may be caused by shearing forces required to break the cell wall. Enzyme activity may be lost if there is foaming of the homogenate in the high-speed blender. Osmotic shock protoplasts ruptured in a hypoosmotic medium.
Detergents solubilize the integral membrane proteins by interacting with the phospholipid bilayer.
The separation of subcellular fractions is achieved by centrifugation. Particles of different density, size, and shape sediment at different rate in a centrifugal field. Differential centrifugation Separates particles as a function of size and density A particular centrifugal field is chosen over a period of time Pellet particles of larger mass Sample distributed throughout the medium Subjected to repeated steps Rate-zonal centrifugation Centrifugation medium is characterized by a positive increment in density density gradient . Sample is applied on the top of density gradient as a narrow band. Particles separate into a series of bands in accordance to centrifugal field, size and shape of the particle and difference in density between the particle and the suspending medium.