Highly Specific Enzyme and Antibody Assays Can Detect Individual Proteins

The purification of a protein, or any other molecule, requires a specific assay that can detect the molecule of interest in column fractions or gel bands. An assay capitalizes on some highly distinctive characteristic of a protein: the ability to bind a particular ligand, to catalyze a particular reaction, or to be recognized by a specific antibody. An assay must also be simple and fast to minimize errors and the possibility that the protein of interest becomes denatured or degraded while the assay is performed. The goal of any purification scheme is to isolate sufficient amounts of a given protein for study; thus a useful assay must also be sensitive enough that only a small proportion of the available material is consumed. Many common protein assays require just from 10-9 to 10-12 g of material.

Chromogenic and Light-Emitting Enzyme Reactions Many assays are tailored to detect some functional aspect of a protein. For example, enzyme assays are based on the ability to detect the loss of substrate or the formation of product. Some enzyme assays utilize chromogenic substrates, which change color in the course of the reaction. (Some substrates are naturally chromogenic; if they are not, they can be linked to a chromogenic molecule.) Because of the specificity of an enzyme for its substrate, only samples that contain the enzyme will change color in the presence of a chromogenic substrate and other required reaction components; the rate of the reaction provides a measure of the quantity of enzyme present.

Such chromogenic enzymes can also be fused or chemically linked to an antibody and used to "report" the presence or location of the antigen. Alternatively, luciferase, an enzyme present in fireflies and some bacteria, can be linked to an antibody. In the presence of ATP and luciferin, luciferase catalyzes a light-emitting reaction. In either case, after the antibody binds to the protein of interest, substrates of the linked enzyme are added and the appearance of color or

Electrophoresis/transfer

Antibody detection

Chromogenic detection

Electrophoresis/transfer

Antibody detection

SDS-polyacrylamide gel Membrane

Incubate with

wash excess

Incubate with enzyme-linked Ab2 ( ); wash excess

Chromogenic detection

React with substrate for Ab2-linked enzyme

SDS-polyacrylamide gel Membrane

Incubate with

wash excess

Incubate with enzyme-linked Ab2 ( ); wash excess

React with substrate for Ab2-linked enzyme

▲ EXPERIMENTAL FIGURE 3-35 Western blotting (immunoblotting) combines several techniques to resolve and detect a specific protein. Step 1: After a protein mixture has been electrophoresed through an SDS gel, the separated bands are transferred (blotted) from the gel onto a porous membrane. Step 2: The membrane is flooded with a solution of antibody (Ab- specific for the desired protein. Only the band containing this protein binds the antibody, forming a layer of antibody molecules (although their position cannot be seen at this point). After sufficient time for binding, the membrane is washed to remove unbound Ab-^ Step 3: The membrane is incubated with a second antibody (Ab2) that binds to the bound Abv This second antibody is covalently linked to alkaline phosphatase, which catalyzes a chromogenic reaction. Step 4: Finally, the substrate is added and a deep purple precipitate forms, marking the band containing the desired protein.

emitted light is monitored. A variation of this technique, particularly useful in detecting specific proteins within living cells, makes use of green fluorescent protein (GFP), a naturally fluorescent protein found in jellyfish (see Figure 5-46).

Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the superior resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. Called Western blotting, or immunoblotting, this multistep procedure is commonly used to separate proteins and then identify a specific protein of interest. As shown in Figure 3-35, two different antibodies are used in this method, one specific for the desired protein and the other linked to a reporter enzyme.

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