Proteins perform many of the critical functions associated with everyday existence for any cell or organism. To best understand how and when and where a specific protein performs its specific functions (whether that be as an enzyme, a structural protein, or even a cell membrane receptor molecule), and what might make that protein change its behavior, requires the ability to identify that one protein in a complex mixture of other proteins.
The ability to specifically detect a given protein has become nearly routine because of techniques that allow for the generation and purification of antibody molecules from mice, rabbits, goats, sheep, and a host of other species, that will recognize a specific protein alone. Using highly specific antibodies, techniques such as Western blotting have been developed to allow for the ready detection of any given protein.
Separating Proteins by Size
To begin characterizing a protein, it is often useful to be able to separate it from as many other proteins in the mixture as possible. One of the first techniques that can be used is to separate it according to how big it is, otherwise known as its molecular weight. When extracts are prepared from a cell, detergents can be used to make the proteins unfold into the linear chain of amino acids. Using a detergent such as sodium dodecyl sulfate (SDS) makes proteins unfold and gives them a net negative charge that is directly related to their molecular size. Using this feature, Dr. U. K. Laemmli developed a system of gel electrophoresis through a plastic medium (polyacrylamide) that easily allowed for proteins to be separated by an electric current strictly according to their overall mass. (1)
Transferring Proteins Out of the Gel Matrix
In order to most easily detect a specific protein, once it has been size fractionated by polyacrylamide gel electrophoresis, requires getting the protein out of the plastic matrix without losing the resolution that was gained by separating it by size in the first place. Towbin et al. (2) solved this problem by developing a technique where the protein mixtures were separated through a planar "slab" gel and then a sheet of a specific type of membrane filter material was placed against the slab gel and electric current was used to force the proteins out of the plastic where they would stick tightly to the membrane filter. This created an exact replica of the pattern of separated proteins, but now the proteins were located on an easily handled piece of filter paper.
Using Antibodies for Detection of Proteins
With the separated proteins immobilized on the filter paper, the speific protein in question can now be detected using antibody molecules that recognize it and it alone. The filter is incubated in a mixture containing the specific antibody (the primary antibody), allowing it to bind to the specific protein. After washing away unbound material, the filter is incubated with another antibody (a secondary antibody) that will detect the the first antibody molecule based on which characteristics it carries from the species in which it was generated. The secondary antibody will also be synthetically labeled with any of a number of compounds that will allow for it to be identified by means including enzymatic, colorimetric or even luminescent. When a protein is separated by gel electrophoresis, and subsequently detected by antibody, it is known as a Western blot.
1 - Laemmli U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature; 227:680-685.
2 - Towbin H., Staehelin T., and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA; 76:4350-4354.
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