Methods for Separating and Identifying Proteins

2 METHODS FOR SEPARATING AND IDENTIFYING PROTEINS 10.2.1 pI-Based Methods of Separation 

An important issue in 2D liquid separations is finding a first dimension, which can provide information on the p7 of the protein. This is important as p7 information has biological significance in proteomics where it is a physical property listed in the databases and can aid in protein identification. The use of p7 

Liquid CF fractions -20 Liquid CF fractions ( Liquid fractions in 

FIGURE 10.1 Schematic overview of protein separation techniques. 

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Since the early 1990s, analytical methods to study protein expression levels and identify proteins have improved tremendously, especially with the development of better separation methods and sensitive mass spectrometric techniques. These methods work well for organisms whose genomes are known, but not as well for non-traditional models, where at best, only limited protein sequence information may exist. For these models, it will be important to develop more robust and high-throughput de novo sequencing methods by mass spectrometry. 

Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the superior resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. Called Western blotting, or immunoblotting, this multistep procedure is commonly used to separate proteins and then identify a specific protein of interest. As shown in Figure 3-35, two different antibodies are used in this method, one specific for the desired protein and the other linked to a reporter enzyme. 

The average composition of copolymers can be determined most readily for the case where the monomeric units can be isolated and identified by suitable scission or degradation reactions. This is the usual method for the elucidation of protein structure. The proteins are hydrolyzed by acids and / or bases in an automated apparatus. The resulting amino acids are chromato-graphically separated and assayed quantitatively via the color reaction with ninhydrine in so-called amino acid analyzers. 

Fractionation of PSII membranes into discrete subsets. The methods of lEF and immunoblotting give us the possibility to separate and identifie chlorophyll-binding proteins both in native conditions, usefull for the study of protein—protein interactions and fully denaturing conditions indispensable when the quantitative relations between apoproteins must be determined. These... 

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