Overview of dialysis, desalting, buffer exchange and protein concentration

Historically, mechanical disruption has been used to lyse cells and tissues; however, detergent-based solutions have more recently been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical disruption. During the protein sample preparation process, many detergents, salts and other molecules used in or generated during protein extraction or subsequent purification may have adverse effects on protein function or stability, or interfere with downstream applications; therefore it may be necessary to remove or reduce these contaminants using one or more protein cleanup methods. Choice of a particular protein clean-up method depends on the sample type and volume, the characteristics of the protein (native or recombinant) and the proposed downstream applications which may include crystallography, mass spectrometry, protein labeling, and other applications. 

General protein extraction workflow. The protocol for total protein extraction from mammalian cells consists of culturing and harvesting the cells, pipetting the sample into clean micro centrifuge tubes, and immediately placing on ice. The tube is then centrifuged to pellet the cells and the supernatant is aspirated. Physical disruption of the cells or the addition of cell lysis buffer is followed by incubation on ice. Finally, samples are centrifuged to collect the supernatant, which contains the total cell proteins.

Dialysis is a classic separation technique that facilitates the removal of small, unwanted compounds from macromolecules in solution by selective diffusion through a semi-permeable membrane. The molecular-weight cutoff (MWCO) of the membrane is determined by the size of its pores. A sample and a buffer solution (called the dialysate, usually 200 to 500 times the volume of the sample) are placed on opposite sides of the membrane. Sample molecules that are larger than the membrane pores are retained on the sample side of the membrane, but small molecules diffuse freely through the membrane and approach an equilibrium concentration with the entire dialysate volume. In this way, the concentration of small contaminants in the sample can be decreased to acceptable or negligible levels.

How dialysis membranes work. A dialysis membrane is a semi-permeable film (usually a sheet of regenerated cellulose) containing various sized pores. Molecules larger than the pores cannot pass through the membrane but small molecules can do so freely. In this manner, dialysis may be used to perform purification or buffer exchange for samples containing macromolecules.

Desalting refers to the removal of salts from a sample, while buffer exchange refers to the replacement of one set of buffer salts with another set. Both goals are easily accomplished by size exclusion chromatography (SEC), also called, gel filtration chromatography. Desalting is accomplished by first equilibrating the chromatography column with water. Buffer exchange, however, is performed by first equilibrating with the column resin with the buffer the sample should end up in. In both cases, the buffer constituents carrying the sample into the column will be replaced by the solution with which the column is pre-equilibrated. 

Both desalting and buffer exchange are separation processes based on gel filtration, also known as molecular sieve chromatography. In this method, a solution containing macromolecules is passed through a column that is packed with a porous resin. When matched correctly, the macromolecules will be too large to enter the pores of the resin and will quickly pass through the column. In contrast, buffer salts and other small molecules will enter the pores of the resin, slowing their rate of migration through the resin bed. This reduction in flow rate causes the faster macromolecules to become separated from the slower, smaller molecules. By collecting separate fractions as they emerge from the column, the macromolecule of interest can be recovered separate from the small molecules which exit the column later. 

Because the solution carrying the sample into the column displaces the solution the resin is equilibrated in, the macromolecules that emerge from the column will be carried in the equilibration buffer. The original buffer is left in the resin, hence the term, buffer exchange.

A number of commercially available desalting devise options exist. For example, Thermo Scientific Zeba products contain resins with exceptional desalting and protein-recovery characteristics. Several Zeba column formats are available for processing sample volumes between 2 µL and 4 mL.

Diafiltration, similar to dialysis, uses a semi-permeable membrane to separate macromolecules from low molecular-weight compounds. Unlike dialysis, which relies on passive diffusion, diafiltration involves forcing solutions through the membrane by pressure (i.e., reverse osmosis, syringe tip sterilization cartridges) or centrifugation. A variety of different types of protein concentrators are commercially available.

During diafiltration, both water (solvent) and low molecular-weight solutes are forced through the membrane-filter where they are collected on the other side. Macromolecules remain on the sample side of the membrane, where they become concentrated to a smaller volume as the water is forced across the membrane to the opposite side. Consequently, typical diafiltration devices that involve centrifugation are called concentrators, and the technique is used primarily for concentrating samples rather than buffer exchange.

Comparison of protein recovery using concentrators from various suppliers. Protein sample solutions were centrifuged in Thermo Scientific Protein Concentrators and other suppliers' concentrators according to manufacturer instructions (20 mL (4,700 x g)). Samples were centrifuged until a greater than 15 to 30-fold decrease in sample volume was achieved; protein concentration was measured by absorbance at A280.

Common protein assay methods depend on measurable color development as a result of chemical reactions or interactions of assay reagents with protein functional groups. However, color development and accurate protein measurement by even the most popular assay methods are sensitive to particular interfering substances that may be present in the samples. For example, most detergents interfere with accurate protein quantitation using Coomassie dye based assays, and reducing agents interfere with the BCA protein assay. One method used to remove interfering substances is to selectively precipitate proteins using trichloroacetic acid (TCA) or acetone. The solution containing the interfering substance is removed and the protein is then resolubilized in an assay-compatible buffer. Commercially available kits simplify sample pretreatment for protein assays. Small molecules may be separated from large proteins in a sample via precipitation with acetonitrile. When used in combination with 96-well centrifuge filter plates, this method is ideal for processing many samples at once.

Protocol for removing substances that interfere with colormetric protein assays. In four steps, the Thermo Scientific Compat-Able Protein Assay Preparation Regent Set removes salts, detergents, reducing agents and other substances from protein samples to eliminate interference with protein assays. This reagent set provides more consistent results compared with homemade TCA or acetone precipitation reagents.

Another general method for protein purification or enrichment is ion exchange chromatography. In ion exchange chromatography, a sample is passed through a charged column. Charged groups on the surface of a protein interact with oppositely charged groups immobilized on the ion exchange support. Ion exchange properties are based on the isoelectric point (pI) of a protein. When a protein is in a buffer with a pH higher than its pI, the protein will have a negative net charge and will bind to a positively charged support or anion exchange medium. When the buffer has a pH below the protein pI, the protein will have a positive net charge and bind to a negatively charged support or cation exchange medium. Changing the pH of the binding buffer will allow for elution of the bound protein of interest. Current Protocols in Protein Science (1990). Supp. 8.4, John Wiley & Sons, Inc., provides an in-depth discussion 

Affinity purification can be used to purify a specific kind of molecule (positive selection) or to remove a specific kind of contaminant (negative selection). Both methods often involve buffer exchange. Affinity purification involves the separation of molecules in solution (mobile phase) based on differences in binding interaction with a ligand that is immobilized by attachment to a stationary support (solid phase). The support or matrix in affinity purification is any material to which a biospecific ligand is covalently attached. Typically, the material used as an affinity matrix is insoluble in the system in which the target molecule is found. Usually, but not always, the insoluble matrix is a solid. Hundreds of substances have been described and utilized as affinity matrices, including agarose, cellulose, dextran, polyacrylamide, latex and controlled pore glass. Useful affinity supports are those with a high surface-area to volume ratio, chemical groups that are easily modified for covalent attachment of ligands, minimal nonspecific binding properties, good flow characteristics and mechanical and chemical stability.

Affinity chromatography (AC). Affinity chromatography is based on the reversible interaction between the target protein, or a group of proteins, and a specific ligand immobilized on a chromatography resin. The process is exquisitely selective and provides high resolution with an intermediate to high sample loading capacity. The protein of interest is tightly bound to the resin under conditions that favor specific binding to the ligand, and unbound contaminants are washed off. The bound protein is then recovered in a highly purified form by changing conditions to favor elution. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity. The target protein is eluted in a purified and concentrated form. 

Separation and analysis of proteins by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is a common laboratory procedure. However, many substances interfere with SDS-PAGE analysis. Commercially available products help speed up sample processing for SDS-PAGE analysis of samples containing interfering substances. Many are able to quickly remove a wide variety of compounds including high concentrations of salts, guanidine, urea and nonionic detergents. The Thermo Scientific SDS-PAGE Sample Prep Kit contains a proprietary resin that binds proteins in the presence of an organic phase. Any interfering contaminants are washed away and proteins are then eluted in a buffer that is compatible with the BCA Protein Assay, allowing quantitation of a portion of the processed sample.

Removing detergents increases resolution of protein separation. Detergent-containing, membrane (m) and soluble (s) protein fractions resulting from extraction with the Thermo Scientific Mem-PER Kit (Part No. 89826) were separated by SDS-PAGE with and without prior treatment with the SDS-PAGE Sample Prep Kit. Image is a chemiluminescent western blot for cytochrome oxidase subunit 4 (COX 4). Kit-treated samples remove the detergent that interferes with electrophoresis while retaining relative protein levels of the original samples. 

Walker JM (2009). The Protein Protocols Handbook. Third Edition. Springer-Verlag New York, LLC.

Asenjo JA, Andrews BA. (2009) Protein Purification using Chromatography: Selection of type, modeling and optimization of operating conditions. J Mol Recognit. 22(2):65-76.