DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 5

the sample is being centrifuged), its molecular mass can be calculated
(independent of the shape of the molecule) with accuracy, usually within
one or two percent.
Sucrose density gradients
Sucrose density gradients formed in an ultracentrifuge can be used
to resolve proteins of different sizes in cell lysates or to resolve different
polymeric forms of a molecule that are produced, for example, during
the synthesis and assembly of the molecule in the cell. This provides im-
portant insights into the biosynthetic intermediates during the synthesis
of complex multimeric proteins. Because sucrose density gradients are
non-denaturing, they will resolve polymeric proteins whether they are
covalently or non-covalently associated. They are therefore very use-
ful as one means of identifying interacting proteins (see protein-protein
interactions below).
Sedimentation velocity
Sedimentation velocity is a common application of gradients. It mea-
sures the rate at which macromolecules move in response to centrifugal
force. The rate of sedimentation provides information about the molec-
ular mass and the shape of the molecule. Velocity sedimentation exper-
iments are carried out at high centrifugal force for short periods of time,
usually only a few hours. Under the conditions used, all macromolecules
will “pellet” if centrifuged for longer periods of time.
Subcellular fractionation
The use of the ultracentrifuge and gradient separation techniques
is not only applied to the study of proteins, but is also widely applied
to subcelluiar fractionation studies, in which different cellular compart-
ments (endosomes, lysosomes, mitochondria, endoplasmic reticulum,
etc.) are resolved. These can be separated by methods that resolve
according to size (velocity sedimentation) or density (density gradient
separation). While sucrose gradients are often used for these studies,
synthetic high density polymers such as Percoll and Nycodenz are now
more widely. This is a useful tool, in conjunction with microscopy, to
identify the subcelluiar compartments in which proteins reside.
Percoll is also used to separate different types of white blood cells
based on their distinct densities.