Protein sample preparation
Centrifugation removes most particulate matter, such as cell debris.
- For small sample volumes or proteins that adsorb to filters, centrifuge at 15 000 × g for 15 min.
- For cell lysates, centrifuge at 40 000 to 50 000 × g for 30 min
If the sample is still not clear after centrifugation, a filtration step can be included. For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium:
|Nominal pore size of filter
||Particle size of chromatographic medium|
||90 μm and greater
||30 or 34 μm
||3, 10, 15 μm, or when extra-clean samples or sterile filtration is required
Samples such as serum can be filtered through glass wool after centrifugation to remove any remaining lipids.
Fractional precipitation is occasionally to remove gross impurities from small sample volumes. Ammonium sulfate precipitation, for example, is frequently used for initial sample concentration and cleanup. The principle of the method is to increase the concentration of ammonium sulfate to a level where proteins begin to “salt out”.
Different proteins salt out at different concentrations, a feature that can be used with advantage to remove contaminating proteins from the crude extract. This method is particularly effective for removing the bulk of albumin, transferrin, and other highly soluble impurities.
Desalting and buffer exchange
Desalting at laboratory scale is a well-proven, simple, and fast method that will rapidly remove low molecular weight impurities and at the same time transfer the sample into the desired buffer in a single step.
Desalting or buffer exchange can be used as a first chromatography step or, when needed, between purification steps for sample conditioning