Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Chemistry of Proteins
Methods for Protein Isolation and Purification
Protein Extraction

Modern tissue disruption Methods are typically combined with the simultaneous extraction of Proteins from tissue homogenates. Most tissue proteins are readily soluble in 8–10% salt solutions. Protein extraction widely employs various buffer mixtures with specific pH values, organic Solvents, and non-ionic detergents—substances that disrupt hydrophobic interactions between proteins and Lipids, as well as between protein molecules.

Among Organic compounds, aside from aqueous glycerol solutions used for a long time, weak sucrose solutions are widely utilized (particularly for solubilization). The pH of the medium significantly affects Protein solubility during extraction; therefore, Protein Chemistry relies on phosphate, citrate, and borate buffer systems spanning acidic to weakly alkaline pH ranges, which facilitate both protein dissolution and stabilization. Tris-buffer systems have become especially widespread; they are mixtures of a 0.2 M solution of tris(hydroxymethyl)aminomethane (HOCH₂)₃CNH₂ (abbreviated as “Tris”) and a 0.1 M Hydrochloric acid solution in varying proportions. Blood Serum proteins are isolated using precipitation methods with ethanol (see Cohn’s method), acetone, butanol, or combinations thereof. Almost all organic solvents break protein-lipid bonds, promoting more efficient protein extraction.

To obtain pure, homogeneous proteins from biological material, various detergents are used to facilitate the Cleavage of protein-lipid complexes and the disruption of protein-protein bonds*. Specifically, Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate are employed to release proteins (Enzymes) tightly bound to the Biomembranes of Cell/35.html">Mitochondria or other subcellular structures.

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* This refers to the disruption of so-called weak bonds (see below). Peptide and other covalent bonds in protein molecules are preserved whenever possible.

Fig. 1.1. Laboratory equipment.

a - handheld pestle homogenizer: 1 - pestle, 2 - body, 3 - motor, 4 - stand; mechanical homogenizer (b) and ball mill (c): 1 - housing with electric motor and starter, 2 - material grinding chamber.

It should be borne in mind, however, that detergents, by inducing the cleavage of protein-protein bonds, disrupt the oligomeric (quaternary) Structure of proteins.