Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

ANALYZING PROTEINS
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(A)
(B)
gel onto a sheet of nitrocellulose paper or nylon membrane. Placing the membrane
over the gel and driving the proteins out of the gel with a strong electric
current transfers the protein onto the membrane. The membrane is then soaked
in a solution of labeled antibody to reveal the protein of interest. This method of
detecting proteins is called Western blotting, or immunoblotting (Figure 8–17).
Sensitive Western-blotting methods can detect very small amounts of a specific
protein (1 nanogram or less) in a total cell extract or some other heterogeneous
protein mixture. The method can be very useful when assessing the amounts of a
specific protein in the cell or when measuring changes MBoC6 m8.20/8.19
in those amounts under
various conditions.
Hydrodynamic Measurements Reveal the Size and Shape of a
Protein Complex
Most proteins in a cell act as part of larger complexes, and knowledge of the size
and shape of these complexes often leads to insights regarding their function. This
information can be obtained in several important ways. Sometimes, a complex
can be directly visualized using electron microscopy, as described in Chapter 9.
A complementary approach relies on the hydrodynamic properties of a complex;
that is, its behavior as it moves through a liquid medium. Usually, two separate
measurements are made. One measure is the velocity of a complex as it moves
under the influence of a centrifugal field produced by an ultracentrifuge (see Figure
8–7A). The sedimentation coefficient (or S value) obtained depends on both
the size and the shape of the complex and does not, by itself, convey especially
useful information. However, once a second hydrodynamic measurement is performed—by
charting the migration of a complex through a gel-filtration chromatography
column (see Figure 8–9B)—both the approximate shape of a complex
and its molecular weight can be calculated.
Molecular weight can also be determined more directly by using an analytical
ultracentrifuge, a complex device that allows protein absorbance measurements
to be made on a sample while it is subjected to centrifugal forces. In this approach,
the sample is centrifuged until it reaches equilibrium, where the centrifugal force
on a protein complex exactly balances its tendency to diffuse away. Because this
balancing point is dependent on a complex’s molecular weight but not on its particular
shape, the molecular weight can be directly calculated.
Figure 8–17 Western blotting. All
the proteins from dividing tobacco
cells in culture were first separated by
two-dimensional polyacrylamide-gel
electrophoresis. In (A), the positions of
the proteins are revealed by a sensitive
protein stain. In (B), the separated proteins
on an identical gel were then transferred
to a sheet of nitrocellulose and exposed
to an antibody that recognizes only those
proteins that are phosphorylated on
threonine residues during mitosis. The
positions of the few proteins that are
recognized by this antibody are revealed by
an enzyme-linked second antibody. (From
J.A. Traas et al., Plant J. 2:723–732, 1992.
With permission from Blackwell Publishing.)
Mass Spectrometry Provides a Highly Sensitive Method for
Identifying Unknown Proteins
A frequent problem in cell biology and biochemistry is the identification of a protein
or collection of proteins that has been obtained by one of the purification procedures
discussed in the preceding pages. Because the genome sequences of most
experimental organisms are now known, catalogs of all the proteins produced in
those organisms are available. The task of identifying an unknown protein (or collection
of unknown proteins) thus reduces to matching some of the amino acid