Immunoblotting Page 1 Immunoblotting of β-Actin Gel electrophoresis is a powerful tool for studying cellular proteins, but has certain limitations. For example, most proteins are not abundant enough within a tissue to be visible when a gel or electroblotted membrane is stained for total protein. A typical mammalian cell possesses thousands of different proteins, and staining will show bands for only the most abundant (a few dozen) proteins (the other proteins appear as a faint blue blur in the background). Furthermore, identifying which band corresponds with a particular protein is rarely possible without further information about the protein (e.g., its molecular weight) or a method of specifically staining that protein. When SDS-PAGE is employed, identifying specific proteins is particularly difficult since SDS denatures the proteins, destroying the native structures and biological activities. Through the technique called Immunoblotting, or Western blotting, a sample separated by electrophoresis is "probed" for the presence of a specific type of protein. by using antibodies. The antibodies are generally prepared in an animal such as rat or mouse, and will bind, ideally, to a single type of protein, even if that protein has been denatured during SDS-PAGE! The results of the immuoblotting are highly specific and very sensitive--a nanogram quantities of a protein can be detected, an amount far too low to detect by staining. Immunoblotting can be used to study the distribution and levels of a protein in different cells, tissues and organs of an organism. The technique has been used to show the presence of similar proteins in different species, since antibodies that bind to a protein from one species may also bind to the same protein from another species. This technique has been widely used in biomedical fields as a means to detect the proteins of pathogens such as HIV in blood, and to identify abnormal proteins in cancerous cells. In this lab exercise, you will use antibodies to probe your nitrocellulose blots with an antibody specific for β-actin, to examine differences in the amount of this protein in liver, muscle and B-16 melanoma cells. Summary of the immunoblotting procedure 1. The protein sample is subjected to gel electrophoresis and electroblotted to a nitrocellulose membrane, and Ablocked@ with a protein solution to saturate any remaining protein binding sites. 2. The nitrocellulose membrane is incubated with a solution of primary (1O) antibody to Aprobe@ for the location of antigenic proteins. We will use anti-β-actin as the 1O antibody. The membrane is then washed free of primary antibody that is not bound to the antigenic protein. 3. The membrane is then incubated with a solution of a secondary (2O) antibody-enzyme conjugate, which binds to the primary antibody. The membrane is then washed free of any secondary antibody conjugate not bound to the primary antibody. 4. The location on the membrane of the secondary antibody conjugate (and thus, also the location of the primary antibody and the antigenic protein) is determined by adding a reagent with which the enzyme of the 2O antibody conjugate reacts to stain the membrane.Immunoblotting Page 2 Figure 1. 2O antibody conjugate. Why is a nitrocellulose membrane “blocked” prior to immunoblotting? As discussed earlier, nitrocellulose binds proteins tenaciously. Antibodies are proteins also, and like all proteins they will bind to nitrocellulose; however, we want our primary antibody to bind only to the antigenic protein on the membrane. To prevent "non-specific" binding of the antibody to the membrane, we >block= (saturate) all of the other binding sites by soaking the membrane in a solution that contains a high concentration of protein. After blocking the nitrocellulose, the primary antibody will only be able to bind to antigenic proteins that have been electroblotted to the membrane. How are antibodies against a specific protein produced? Animals can be immunized artificially to produce antibodies that bind to specific types of antigens. This is how antibodies are produced for use in Western blotting. To induce antibody production, a laboratory animal, such as a mouse, is injected with the protein against which antibodies are needed. The protein acts as an antigen, and, over a period of a few weeks, antibodies that bind to that protein will begin to increase in the animal's blood. Eventually, samples of the animal's blood serum containing the antibodies can be collected and used for Western blotting. What is the difference between 1O and 2O antibodies? In the immunoblot procedure, two types of antibodies are used. Primary (1O) antibodies are the antibodies that bind specifically to the protein being studied.. A 1O antibody might be referred to "rabbit-anti-β-actin antibody@ (for example), which means it was created by injecting purified β-actin into a rabbit. The primary antibody is used to probe (search) the nitrocellulose membrane for antigenic proteins. However, binding of the primary antibody will not make visible the position of the protein band B this requires probing the nitrocellulose membrane again with a secondary (2O) antibody conjugate. Secondary antibodies have two important features (Figure 1); they bind to other antibodies and they are covalently conjugated (linked) to an enzyme, such as peroxidase or alkaline phosphatase. The conjugated enzyme provides a means to identify the position on the nitrocellulose of the target protein. A typical 2O antibody might be referred to as Agoat-anti-rabbit IgGBalkaline-phosphatase-conjugate@ (for example), i.e., an antibody produced in goats that binds to the mouse antibody type IgG and which is coupled to the enzyme ‘alkaline phosphatase’. When the nitrocellulose membrane is Aprobed@ with a solution of secondary antibodies, they will bind to the primary antibodies (Figure 2). The activity of the conjugated enzyme can then be used to reveal the position on the nitrocellulose of the secondary antibody. The membrane is treated with a reagent that changes from a soluble, colorless form to an insoluble, colored form. For example, alkaline phosphatase converts a chemical from a clear to a bluish-black product that sticks to the membrane. The location of the resultant brown band shows the position of the original protein band. What types of controls should be run?Immunoblotting Page 3 Figure 2. 2O antibody bound