Immunological procedures, protein

Babajimopoulos, M. and Mikolajcik, E. M. 1977. Quantification of selected serum proteins of milk by immunological procedures. J. Dairy Sci. 60, 721-725. 

In addition to analyzing a protein for characteristics dictated by its specific nature, routine analytical assays are performed namely, amino acid analyses and amino- and carboxy-terminal sequencing. If necessary, disulfide assignments are made. A search is ordinarily conducted for oxidations and/or deamidations. In the case of recombinant proteins particular attention is paid to detecting signal sequences or proteolytically degraded species. In contrast, deletions and chemical modifications can be a consequence of proteins prepared by chemical synthesis. Lastly, tests are applied to determine if the protein is correctly folded into its native three dimensional structure. In addition, with recombinant proteins, sensitive immunological procedures are performed to ensure that host cell proteins have not been copurified with the protein of interest. 

Since the identities of the gonadotropic hormones remain uncertain, it is important before discussing assay procedures, to examine in some detail the various techniques for their preparation. Such methods may of themselves lead to differences in the composition or structure of the final products of extraction as well as to variations in the type of protein contaminants remaining in the purified hormone preparation. Since immunological procedures hinge on the availability of pure FSH and LH, it is likely that the results of such assays will vary depending on the methods by which these purified hormones have been obtained. 

After chronic exposure, cadmium accumulates in the human body and causes kidney diseases, especially lesions of proximal tubular cells. A tubular proteinuria causes an increase in urinary excretion of microproteins. Excretions of retinol binding protein (RBP), (32-microglobulin ((32-M), and a 1-microglobulin are validated biomarkers for analyzing cadmium effects. For this purpose, immunological procedures such as ELISA, and radio- and latex-immunoassays are used. 

An extract of the soluble proteins of the bacterium E. coli was provided by the Giddings lab in the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. The details of the procedure for the preparation of this extract have been reported elsewhere (Link, 2004). 

The PAP method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme/antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that... 

Subsequent work by Gardiner et al. showed that in a relatively complex mixture like human serum, the association between a metal and protein or ligand could be said to have been established when their Gaussian distribution coincide. This is more likely to be true if the elution volumes of the constituents are in the fractionation range rather than in the excluded volume. Examples of the usefulness of immunological techniques as an aid in identification of proteins are given. Necessary clean-up procedures are also suggested. 

Immunological studies are not yet widely used in the context of screening procedures, but they are becoming easier to perform [77]. Their use is rendered possible by the observation that at least a subset of the OXPHOS defects originates from, or results in, anomalies in complex assembly with increased protein degradation. As a result, a net decrease of the immunoreactive material can be used to trace some OXPHOS deficiencies. The large number of antibodies available makes this approach... 

Another important procedure is to screen for production of the desired protein by using immunological methods with antibodies raised against it. 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

Lectins, or proteins with specific binding sites for carbohydrates, can be used as targeting molecules to localize particular glycoconjugates such as glycoproteins or glycolipids on cell surfaces (Fig. 373). Labeled with gold particles, lectins are important probes for detection of cell surface components and intracellular receptors and in immunological or biochemical assay procedures (Bog-Hansen et al., 1978 Kimura et al., 1979 Nicolson, 1978 Roth, 1983 Benhamou et al., 1988 Nakajima et al., 1988). 

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