Gel filtration chromatography is often used to perform desalting and buffer exchange with the aid of various size exclusion chromatography resins. Desalting involves the chromatographic separation of macromolecules from smaller molecules such as buffer components, salts and unreacted labeling reagents. Buffer exchange is used to switch a protein or molecule into a more appropriate buffer for applications such as electrophoresis, ion exchange, affinity chromatography or storage.
In both desalting and buffer exchange, the macromolecular components are recovered in the same buffer used to equilibrate the resin within the gel filtration column. If water is used for equilibration, the components will be desalted. If another buffer is used, buffer exchange will result. Buffer exchange may be performed with each of the desalting products listed below after pre-equilibration with the desired buffer.
Zeba Desalting Products
Thermo Scientific Zeba Desalting Products contain a proprietary high-performance size exclusion resin that offers exceptional desalting and protein-recovery characteristics compared to other commercially available resins. Even very dilute (25 µg/mL) protein samples can be successfully processed to obtain greater than 95% retention (removal) of salts and other small molecules and good recovery of proteins and other macromolecules.
The Zeba Spin Desalting Columns (available in five column sizes) and two MWCOs (7K and 40K) provide an easy-to-use format that dramatically improves results over standard drip-column methodologies. This eliminates the need to wait for samples to emerge by gravity flow and monitor fractions for protein recovery. Zeba Desalting Columns require no chromatography instrumentation, cumbersome column preparation or equilibration and they can process multiple samples in approx. 8 minutes.
Zeba Desalting Resin also comes pre-packed in 96-well filter/centrifuge plates and in two sizes of Pierce Chromatography Cartridges.
Zeba Desalting Resin Products and recommended sample volumes
| 7K MWCO | 40K MWCO | Resin bed | Sample size |
|---|---|---|---|
| Micro spin column | Micro spin column | 0.075 mL | 2 to 12 µL |
| 0.5 mL spin column | 0.5 mL spin column | 0.5 mL | 30 to 130 µL |
| 2 mL spin column | 2 mL spin column | 2 mL | 200 to 700 µL |
| 5 mL spin column | 5 mL spin column | 5mL | 600 to 2000 µL |
| 10 mL spin column | 10 mL spin column | 10 mL | 1500 to 4000 µL |
| 96-well spin plate | 96-well spin plate | 0.55 mL | 20 to 100 µL |
| 1 mL cartridge | 1 mL | 50 to 250 µL | |
| 5 mL cartridge | 5 mL | 100 to 1500 µL |
Note: Sample size ranges given here are for the 7K MWCO products. Ranges for the 40K MWCO products are slightly different; see the product page for specific values.
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Polyacrylamide size exclusion chromatography columns and spin columns
Thermo Scientific Polyacrylamide Desalting Columns are pre-packed with polyacrylamide size exclusion resin. The 6K MWCO columns have a wet bead diameter of 90 to 180 µm. The 1.8K MWCO columns have a wet bead diameter of 45 to 90 µm. These columns are ideal for separating low MW compounds with a fractionation range of 0.1 to 1.8K. The polyacrylamide size-exclusion resin is not subject to enzymatic degradation and will not serve as a nutrient for microbial growth. The resin is very hydrophilic, so hydrophobic binding to sample molecules is minimized. Polyacrylamide resins also eliminate sample contamination by low MW sugars which can occur when using crosslinked polysaccharide supports. In addition, oxidizing agents can be removed without destroying the support. This resin is susceptible to hydrolysis of amide groups under extreme pH conditions, so an operating pH of 2 to 10 is recommended at room temperature. The polyacrylamide resin can also be autoclaved at pH 5.5 to 6.5 for 30 minutes at 120°C.
Polyacrylamide Desalting Columns and recommended sample volumes
| Column size | Resin bed | Sample size |
|---|---|---|
| 0.7 mL spin column | 0.7 mL | 30 to 120 µL |
| 5 mL gravity column | 5 mL | up to 1.25 mL |
| 10 mL gravity column | 10 mL | up to 2.5 mL |
Note: The optimal sample size for this matrix is 1/10 the column resin bed. However, sample volumes up to 1/4 of the column volume bed may be used if 100% desalting is not required.
Dextran-resin gel filtration chromatography columns
Thermo Scientific Dextran Desalting Columns (5K MWCO) are pre-packed with crosslinked dextran size exclusion resin. The wet bead diameter is 50 to 150 µm. The resin has good rigidity for easy handling and excellent flow properties. The Dextran resin is stable in water, salt solutions, organic solvents and alkaline or weakly acidic solutions. It is heat-stable and can be autoclaved dry or in solution at a neutral pH for 30 minutes at 120°C without affecting its chromatographic properties.
Dextran desalting columns and recommended sample volumes
| Column size | Resin bed | Sample size |
|---|---|---|
| 5 mL gravity column | 5 mL | up to 1.25 mL |
| 10 mL gravity column | 10 mL | up to 2.5 mL |
Note: The optimal sample size for this matrix is 1/10 the column resin bed. However, sample volumes up to 1/4 of the column volume bed may be used if 100% desalting is not required.
For Research Use Only. Not for use in diagnostic procedures.