Specific Proteins Can Be Detected by Blotting with Antibodies
A specific protein can be identified after its fractionation on a polyacrylamide gel by exposing all the proteins present on the gel to a specific antibody that has been labeled with a radioactive isotope or a fluorescent dye. This procedure is normally carried out after transferring all of the separated proteins present in the 100 50 25SDS migration (mol. mass in kilodaltons)
Figure 8–16 Two-dimensional polyacrylamide-gel electrophoresis.
All the proteins in an E. coli bacterial cell are separated in this gel, in which each spot corresponds to a different polypeptide chain. The proteins were first separated on the basis of their isoelectric points by isoelectric focusing in the horizontal dimension. They were then further fractionated according to their molecular mass by electrophoresis from top to bottom in the presence of SDS. Note that different proteins are present in very different amounts. The bacteria were fed with a mixture of radioisotope-labeled amino acids so that all of their proteins were radioactive and could be detected by autoradiography. (Courtesy of Patrick O’Farrell.) 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 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.
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