Overview of Dialysis, Desalting and Buffer Exchange
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.
Dialysis is a separation technique that gained popularity in life science laboratories during the 1950s. Research papers of that era described dialysis as a new, cutting-edge tool that scientists could use to unravel complex mixtures of biomacromolecules. Many of the dialysis theories established at that time are the cornerstones for contemporary products featured in this brochure. There are, however, two major differences between the dialysis tools of yesterday and today—preparation time and the amount of sample loss due to leaks. Early laboratory dialysis methods involved dedicating a significant amount of time to membrane preparation; Thermo Scientific Pierce Dialysis Products are essentially ready to use and resist sample leakage.
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. Desalting is accomplished by first equilibrating the chromatography column with water. Buffer exchange, however, is performed by first equilibrating with the column resin with a 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 a separation process 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.
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. Popular devices include Amicon*, Centricon*, Vivaspin* brands of concentrators.
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.
Common protein assay methods depend on measurable color development as a result of chemical reaction or interaction 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 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 the protein using Trichloroacetic acid (TCA) or acetone. The solution containing the interfering substance is removed and then the protein is resolubilized in an assay-compatible buffer. Commercially available kits are also available to simplify sample pretreatment for protein assay measurement. Small molecules may be separated form large proteins in a sample via precipitation with acetonitrile. Used in combination with 96-well centrifuge filter plates, this method is idea for processing many samples at once.
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 on ion exchange chromatography for researchers interested in more detail on this subject.
Systems for sample preparation involving affinity purification are described in the article called Overview of Affinity Purification. Affinity purification can be used to purify one specific kind of molecule (positive selection) or to remove a specific kind of contaminant (negative selection). Both methods often involve an exchange of the buffer.
Separation and analysis of proteins by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is an essential and common laboratory procedure. However, many substances interfere with SDS-PAGE analysis. Commercially available products are available to speed up sample processing for SDS-PAGE analysis of samples with 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 Sample Prep Kit work via proteins binding to a proprietary support in the presence of an organic phase and are eluted in a buffer that is compatible with the BCA Protein Assay.
For Research Use Only. Not for use in diagnostic procedures.