Western immunoblot technique

Figure 9.7 demonstrates the reduction in impurities with each phase separation cycle. In short, three cycles reduce impurities by over a factor of 1,000. Using Img of polymer, three cycles of phase separation decrease impurities from 1,428,600 parts per billion (ppb) to 1,365 ppb. As discussed below, further purification lowers impurities, as judged by Western immunoblot techniques, to less than 5 ppb. 

Western Immunoblot Technique to Demonstrate Impurity Levels... 

Having demonstrated production of designed protein-based polymers, the next issue is one of achieving purification adequate for the intended application. The most sensitive means with which to detect impurities of relevance to medical applications is the Western immunoblot technique. )Wth this technique, levels of purification are demonstrated in Figure 9.12 for the following model proteins ... 

Figure 9.12. Western immunoblot techniques for placing a limit on the impurity burden of a series of protein-based materials. (Upper plate) Absence of line indicates impurities are less than 1 ppm as achieved by three phase separation cycles. (Lower plate) Absence of dot indicates impurities are less than 5ppb. See text for further discussion. (Reproduced with permission from Urry et al. )...

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

Key Words Western blotting immunoblotting electrophoresis 2D electrophoresis immunoaffinity identification immunoblotting techniques. 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

The technique of immunoblotting, often referred to as western blotting, is frequendy used for a variety of applications where protein concentrations... 

Western blotting A technique for identifying proteins or protein fragments in a mixture that react with a particular antibody. The mixture is first resolved into bands by one-dimensional denaturing gel electrophoresis. The protein bands are then blotted onto a nitrocellulose sheet, the sheet is treated with the antibody and any bands that bind the antibody are identified. More accurately called immunoblotting. 

The western blot, also known as the immunoblot, is used to identify and relatively quantify the amount of a specific protein within a sample containing multiple proteins, such as what is obtained following cell lysis. An immunochemical assay is used for protein identification. The technique is often used to detect the downstream product of specific gene expression. Due to high sensitivity, efficiency, and convenience, the western blot has been widely used by researchers through the world. 

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