Biology Reference
In-Depth Information
9. Freeze/Thaw
The fact that ice occupies a larger volume than does an equal quantity of liquid water
(water is denser than ice, see Chapter 3), can be used to disrupt cells
[23]
. Alternating rapid
freezing in liquid nitrogen or in a slurry of dry ice and isopropanol, followed by thawing in
warm water can homogenize cells. More often, freeze/thaw cycles are used as a preliminary
step to be followed by another homogenization procedure.
C. M
EMBRANE FRACTIONATION: CENTRIFUGAT
ION
By far the major technique that has been employed to separate the various cell fractions is
centrifugation
[8,24
26]
. Two basic types of centrifugation exist, differential and density
e
gradient.
Differential Centrifugation
Differential centrifugation is a first crude step in separating major cell fractions whose
physical properties, size, and density, are very different from one another. In fact at least
one order of magnitude difference in the sedimentation coefficient (discussed below) is
required to achieve separation, and even then separation is not complete.
After the tissue sample is homogenized the mixed cell contents are subjected to repeated
centrifugations at increasing speed, a process known as differential centrifugation. After each
centrifugation the pellet (heavier component) is removed and the remaining cell suspension
(lighter components) is centrifuged at an increased speed. The following separations are
achieved from biggest (heaviest) to smallest (lightest) fractions:
BIGGEST
Pellet 1
unbroken cells
Pellet 2
nucleus
Pellet 3
mitochondria
Pellet 4
lysosome
Pellet 5
microsomes
Pellet 6
ribosomes
SMALLEST
Suspension
cytoplasmic proteins, sugars,
amino acids, nucleotides, salts etc.
Centrifugation speeds in membrane studies vary over a wide range from ~1,000g for 5 min
for unbroken cells up to 1,000,000g for hours to finally pellet cytosolic macromolecules. Each
step in differential centrifugation only
enriches
the target membrane. It does not completely
purify the membrane.
The limitation of differential centrifugation results from large, heavy particles initially
residing at the top of the centrifuge tube pelleting at the same time as smaller, lighter particles
that are initially near the bottom of the tube. For example, consider the case of a mixture of
