Protein separation techniques immunoblotting

The initial characterizations of the various fusion proteins (Subheading 3.1.1, step 7), as well as more detailed biochemical assays (Subheading 3.3) make extensive use of electrophoretic separations and immunoblotting. Protocols used in the authors laboratories for these various techniques (SDS-polyacrylamide gel electrophoresis, native acrylamide gel electrophoresis, and separation of nucleic acid on acrylamide gels containing urea) are described in this section. 

A significant advantage of this method is that is provides quantitative information since the IEF technique actually separates the phospho- and dephospho- forms of the protein, the ratio of these species as detected by IEF/immunoblotting is a direct readout of the protein s phosphorylation status within the cell (assuming no selective loss of one species during isolation and analysis). 

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%,... 

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. 

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

In immunoblotting procedures, a mixture of antigens is first separated by gel electrophoresis and subsequently transferred from the gel to a membrane, where it is detected by antibody. Comparative levels of reactivity with antibody can be assessed by incubation with serially diluted antibody preparations. A comprehensive review of the technique is available.44 Because the test is done with proteins that are at least partially degraded, relatively distant relationships can be detected.43 46 The method has been used to calculate SDI values for proteins of potyviruses.47... 

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. )...

In many cases, for the purpose of protein identification, immunochemical techniques have been combined with separation methods and with other methodologies. A good example of a combined procedure is the identification of the protease-resistant pathological form of the prion protein, in which the products of specific proteolysis of the whole tissue are first separated by electrophoresis, and identified by specific monoclonal antibodies after transfer (blotting) to a suitable membrane. Another common application of immunoblotting techniques is the identification of the food proteins (and of peptides... 

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