Protein removal from samples

Proteins are best removed from sample before injection, and various techniques will be described in the sample preparation section for doing so (Chapter 12). If you must shoot crude sample-containing protein, use a guard column and change it often. A new guard column might be less expensive than your time needed to clean it and, certainly, will be less expensive than a new column. 

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

Sample preparation used to extract proteins from cells prior to analysis is an important step that can have an effect on the accuracy and reproducibility of the results. Proteins isolated from bacterial cells will have co-extracted contaminants such as lipids, polysaccharides, and nucleic acids. In addition various organic salts, buffers, detergents, surfactants, and preservatives may have been added to aid in protein extraction or to retain enzymatic or biological activity of the proteins. The presence of these extraneous materials can significantly impede or affect the reproducibility of analysis if they are not removed prior to analysis. 

The phosphorescence lifetimes have been examined for many protein systems as a function of temperature. In the early work oxygen was not removed from the sample.(72,73) In these works the lifetimes are dominated by quenching by oxygen, and so the temperature dependencies probably represent temperature-dependent oxygen diffusion. 

Dialysis and ultraflltration have been largely applied to isolate and fractionate food proteins and peptides. To isolate the protein fraction from wine and must samples, different authors used dialysis followed by lyophilization to concentrate the dialyzed samples [106,108,109], Depending on the application, membrane of different material, filtration surface and cut-off, able to fractionate the molecules in function of their molecular size, can be used to remove either proteins and other macromolecules or amino acids and small peptides. 

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