DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 4

While the Bradford assay is easy to carry out and relatively quanti-
tative, the most accurate measure of protein concentration is using a
spectrophotometric approach. This is based on the absorbance of ultra-
violet light at 280 nm by the protein. The side chains of the amino acids
tryptophan and tyrosine are primarily responsible for the absorbance,
with a minor contribution of cystines (disulfide bonds). If the amino acid
composition of the protein is known, the most accurate determination of
protein concentration can be obtained with this method.
Gels and gradients for separation
of macromolecules
The size of biomolecules such as proteins or a piece of DNA can be
determined by separation on gradients or gels. For gradient separation,
sucrose density gradients are frequently used. The gradient forms in a
tube when the sucrose mixture is subjected to a high centrifugal force
using an ultracentrifuge. The larger macromolecules migrate farther in
the gradient, while smaller ones migrate less, giving a degree of sep-
aration that varies given the differences in size of the macromolecules
being separated, the concentration of the sucrose in the gradient, and
the complexity of the starting material. One of the major advantages of
sucrose gradient separation is that it does not denature the proteins and
thus, non-covalently bound proteins can be isolated as protein-protein
complexes. The development of the ultracentrifuge in the early twenti-
eth century, and their use to determine precise molecular weights, was
a major advance in protein chemistry. For his efforts in the development
and use of the ultracentrifuge, Theodor Svedberg won the Nobel Prize
in Physics in 1926. Nevertheless, the ultracentrifuge did not become a
standard tool for separating biomolecules until the late 1940s and later.
Today, ultracentrifuges are manufactured that can generate speeds of
100,000 to 130,000 rpm (revolutions per minute) and g forces (mea-
sured against the force of gravity) of 600,000 × g to 1,000,000 × g,
reducing the time required for centrifugation.
Sedimentation equilibrium
Sedimentation equilibrium is a method for measuring protein molec-
ular masses in solution and for studying protein-protein interactions. It
can be used to determine if the native state of a protein is multimeric
(dimer, trimer, etc.), and determine the stoichiometry of complexes that
form from two or more different proteins. Using an analytical ultracen-
trifuge (which allows the distribution of a protein to be measured while