Peptide immunochemical methods

Fig. 9.2 Relative concentrations of P450 in human liver microsomes. a P450s in 60 liver samples were estimated using immunochemical methods (electrophoresis/immunoblot-ting) [52]. Because of cross-reactivity, the individual P450s in subfamilies are not distinguished. The unknown fraction is the difference between the sum of the immunochemi-cally determined forms and the total amount, calculated from Fe -CO versus Fe " difference spectroscopy [53]. b-d Estimates were made using liquid chromatography-mass spectrometry (LC-MS) proteomic analysis with heavy-atom peptides, b Results of an analysis of 50 pooled human liver samples (XenoTech, HLM610 preparation) [54]. c Results reported in the same reference as Part 5 [54] as means from analysis often individual human samples, d Analysis of a pooled set of 23 human liver samples by another laboratory [55]...

I, and I. Radioimmunoassay combines the specificity of an immunochemical reaction with the sensitivity of isotope analysis (F4, S19) and is currently developing rapidly for the analysis of steroids, peptide hormones, and specific proteins (G9). Enzymes can be determined with labeled substrates (01). The requirements of standardization and dosimetry make it probable that these methods will continue to be based on relatively large automatic instruments. However, the greatest problems are unlikely to be in the counting equipment but in sample handling and processing and in standardization of the system. 

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... 

More localized variations can be detected by immunochemical studies when the antibodies are purified to monospecificity. To obtain information regarding very localized parts of the polypeptide chain during the folding (or the unfolding) process, the most suitable methods are certainly those which allow one to determine the accessibility of amino acid side chains to chemical reagents in the precise position of the sequence, or the accessibility of definite peptide bonds to specific proteases. Nuclear magnetic resonance allows one to follow the behavior of individual protons when correct assignment is possible and thus provides the same type of local... 

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