Protein study techniques

A new approach to study root exudation of distinct compounds in soil-grown plants uses inoculation of roots with genetically engineered reporter bacteria, which are able to indicate the presence of particular compounds by indicator reactions, such as production of ice-nucleation proteins. This technique has been employed to detect the release of amino acids from roots of soil-grown A vena harbata (56). 

Just as gel electrophoresis methodology is commonly used to determine homogeneity, as well as molecular weights of proteins these techniques may be used similarly for dendrimer analysis. Other uses have included the assessment of DNA/dendrimer binding constants which have been studied using gel electrophoresis. Application of gel electrophoresis in dendritic science will be discussed from these three aspects. 

The examples described in this chapter are meant to illustrate the actual application of NMR for the study of protein-ligand interactions in the pharmaceutical industry. In our group, the focus has been mainly on protein observe techniques, as we have been fortunate enough to have a dedicated laboratory for providing both unlabeled and labeled proteins in large quantities. At present, two-dimensional HSQC spectra offer the highest content of information which is not accessible that rapidly by other methods. 2D NMR spectroscopy provides... 

PAG electrophoresis is an important technique in many areas. In DNA sequencing it provides a fundamental method of separation, and in protein studies it is important both in the form described here and also in conjunction with the detergent, sodium dodecyl sulphate (SDS), giving a valuable method for assessing the relative molecular mass of a protein. 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

Because the sequencing of DNA has become so straightforward, genes of several ribosomal proteins have been sequenced to allow an independent determination of the primary sequence of some of the proteins (Post et ai, 1979 Olins and Nomura, 1981). These studies have confirmed the sequences previously elucidated by protein chemical techniques. In the case of SI (the largest of all E. coli proteins), the combination of amino acid- and nucleotide-sequence determinations was used to provide the sequence (Schnier et ai, 1982). 

Morr, C. V., Van Winkle, Q. and Gould, I. A. 1962. Application of polarization of fluorescence technique to protein studies. III. The interaction of x-casein and (3-lacto-globulin. J. Dairy Sci. 45, 823-826. 

The essence of modem work on structure and activity of proteins is analysis at the level of individual residues and atoms. Unfortunately, the only state that is readily, studied at this level is the native state. The only spectroscopic technique that can be applied in solution with sufficient resolution is NMR, but it works best with stable, compactly folded proteins. New techniques are required for the... 

This text covers the design and execution of enzyme assays. Chapters I. 9. and 11 ( Principles of enzyme assay and kinetic studies, Techniques of enzyme extraction," and Buffers and the determination of protein concentration," respectively) are particularly relevant to this unit. 

While the technique of X-ray crystallography is a complex instrumental and computational procedure, it can reveal the structure of very large macromolecules, which means that it permits the determination of the structure of both cytoplasmic proteins and membrane-bound proteins (proteins that are typically drug targets). While X-ray crystallography is very useful, however, it should be emphasized that proteins studied in this manner are in a crystalline state, a state that does not resemble the normal physiological (liquid) environment of the cell or body (Wishart, 2005). 

Oligonucleotide-directed site-specific mutagenesis is a powerful technique for the study of proteins and has brought about great progress in research on the structure and function of proteins. This technique makes it possible in theory to obtain any mutant enzyme desired. One of the goals of such protein engineering is to make the proteins more stable. 

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