Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Concentration of Protein Solutions

To concentrate Proteins from dilute solutions, precipitation with salts, acids, and organic Solvents (acetone, methanol), ultrafiltration, dialysis, evaporation, and lyophilization are employed. These Procedures become increasingly challenging at lower protein concentrations in the solution; for instance, trichloroacetic acid fails to precipitate proteins present at 1–25 µg [17].

Proteins are effectively concentrated using PAGE [63, 196, 276, 277, 382] or isotachophoresis [283]. Electrophoretic techniques for macromolecule concentration via precipitation [308] or Protein Extraction from non-ionic solutions using membranes [3] can be complicated by irreversible protein sorption onto the membrane. This issue is also encountered when working with liquid membranes [384, 385] (see below). Proteins from a dilute solution (100–800 ng/mL) were isolated by precipitation following radioactive labeling with [³H]-1-fluoro-2,4-dinitrobenzene [280]. Carboxymethyl dextran has been used to "strip" proteins from DEAE-Cellulose in a highly concentrated form [367].

Lyophilization is the most widely used method for concentrating protein solutions. The solution is rapidly frozen (using acetone, ethanol, or isopropanol mixed with dry ice), and Water and volatile buffers are removed under the vacuum of an oil pump equipped with a special trap. The trap is filled with alkali when evaporating acidic solutions; concentrated formic acid must be diluted with water to a 30% concentration prior to freezing.

For rapid removal of water, organic solvents, and volatile buffers, a specialized Savant concentrator (Speed-Vac Concentrator) can be used, where evaporation takes place under vacuum in a low-speed centrifuge with Temperature control. The kit includes centrifuge tubes of various volumes. This method is particularly convenient for concentrating fractions from chromatographic columns or sequenators, being both faster and more practical than evaporating individual samples in a stream of nitrogen.

Protein precipitates obtained after extraction with non-polar solvents or high-temperature drying may have poor solubility. If the protein is present as a thin film, it can be dissolved using 1 M NaOH [1 h at room temperature or heating to 100 °C for 10 minutes or more].

Effective protein solvents include aqueous hexafluoroacetone [48, 82] and trifluoroacetic acid [236]. Structured proteins such as Collagen and keratin, upon Treatment with H₂O₂, are readily soluble in 0.05% SDS in IMNaOH at 100 °C (a property utilized in determining the concentration of these proteins via the biuret reaction) [130].

When freeze-drying frozen solutions containing trace amounts of free Amino Acids, short Peptides, or other low-molecular-weight compounds under high vacuum, There is a risk of significant substance loss, making quantitative Assessment of the overall process impossible. For example, it was found that 90 minutes of lyophilization at a vacuum of 5–10 mm Hg (cooled with a mixture of C₂H₅OH + solid CO₂) resulted in the loss of 89% of 2-[¹⁴C]-L-phenylalanine and 86% of N-feruloylglycyl-L-phenylalanine [378].

Dialysis using dialysis tubing (such as Visking) is employed to remove non-volatile buffers, salts, performic acid, and other components from protein solutions. Researchers also have access to a wide range of commercially available cellulose filters designed for molecular weight ranges from 1,000 to 50,000 (Spectrapor membrane, Spectrum Medical Industries Inc.). Protein solutions can also be concentrated using ultrafiltration apparatus. It should be noted that there is a possibility of protein loss in dialysis tubing due to adsorption.

To concentrate peptides dissolved in large volumes of sodium acetate buffer (pH 7.5) containing 4 M urea, reversed-phase columns such as Lichrosorb C8 are used. The eluate containing acetic and formic acid salts is typically monitored using a fluorescence detector. Rapid Desalting from urea and buffer components is achieved using mini-columns [278] or specialized Waters cartridges [16, 392] (see also Section 6.2.3).

A microconcentrator equipped with a low-adsorption Centricon membrane allows for the rapid (30–60 min) concentration of small volumes (2 mL) down to 25–40 µL in a centrifuge with a fixed-angle rotor. Desalting or buffer exchange can be performed simultaneously with concentration (Amicon – Scientific Systems Division).

Since SDS is a denaturing agent, its complete removal is often necessary. Employing Electrophoresis and anion-exchange Chromatography for this purpose results in low protein yields. It is recommended to remove SDS via so-called ion-pair extraction using triethyl- or tributylammonium, which provides high yields (70–100%) across microgram to milligram quantities of protein [153].