Abstract
The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes.
Highlights
This Protocol Article describes the use of differential centrifugation to fractionate a mammalian liver homogenate but similar methods should be applicable to all mammalian tissues, cultured cells, and organisms such as yeast, as long as an efficient homogenization procedure is available
The solutions used for homogenization and for washing and resuspension of the pellets, depends upon the organelle to be purified. They were developed for work with rat liver and other soft tissues and generally contain sucrose as the osmotic balancer
Many cultured cells can be homogenized in the General Purpose medium or some other similar isoosmotic medium[4]
Summary
This Protocol Article describes the use of differential centrifugation to fractionate a mammalian liver homogenate but similar methods should be applicable to all mammalian tissues, cultured cells, and organisms such as yeast, as long as an efficient homogenization procedure is available. High-speed centrifuge with fixed-angle rotor to hold 30- to 40-ml polycarbonate tubes (see Notes 1 and 2) 3. Pellet the nuclear fraction by centrifugation at 1,000gav for 10 min
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