What is the Role of Buffer System in Protein Extraction and Clarification?
During the protein clarification and extraction process, the proteins under examination can easily become unfolded, denatured, or damaged, often before they’ve been clearly identified. Proteins may also separate from the assay solution or become entangled with non-relevant cell components like lipids and DNA.
Choosing a Buffer System for Protein Extraction: Considerations
To accomplish this goal, researchers need to choose a buffer solution that’s compatible with the protein in question and recreates an ionic environment similar to the ionic environment of the cell. The pH balance of the buffer must correspond with that of the cell in vivo, while still allowing researchers to separate the cell’s component parts.
To keep an extraction timely and efficient, and to avoid the need to switch buffer solutions during the process, it’s wise to choose a buffer solution that can maintain protein stability during every stage of the procedure, including chromatography or electrophoresis. It’s also wise to account for shifts in temperature, since the pH of some buffer solutions can change in the presence of heat.
A few widely compatible and therefore commonly used buffer solutions include Tris-HCL, HEPES-NaOH, and sodium dihydrogen phosphate - disodium hydrogen phosphate. These buffers are not ideal for all solutions, however. Tris buffers, for example, have a pH that can be strongly affected by temperature and concentration. And HEPES buffers should not be used with the Lowry Assay or any studies involving radicals, since they can form radicals under various conditions.