Extraction Buffer
Extraction buffer
Extraction buffers, also sometimes referred to as lysis buffers, are a buffer solution that is used to break open cells for use in molecular biology experiments that analyze compounds in cells. Most lysis buffers contain salts to regulate the acidity and osmolarity of the lysate.
- Typically, extraction buffers have an ionic strength (0.1–0.2 M) and a pH (7.0–8.0) that is considered compatible with that found within the cell.
- Tris or phosphate buffers are the most widely used.
- Each buffer option has a specific pH range, so the buffer should be chosen based on whether the target substance is stable under a certain pH.
- Also, for buffers with similar pH ranges, it is important to consider whether the buffer is compatible with the target constituent of the cell.
Components of an extraction buffer
A variety of other reagents can be included in the buffer for specific purposes. These include:
1. An antioxidant
- Inside the cell, the protein is in a highly reducing environment, but when it is released into the buffer, it is exposed to a more oxidizing environment.
- Since most proteins contain several free sulfhydryl groups (from the amino acid cysteine), these can undergo oxidation to give inter and intramolecular disulfide bridges.
- To avoid this, reducing agents such as dithiothreitol, b-mercaptoethanol, cysteine, or reduced glutathione is often included in the buffer.
2. Enzyme inhibitors
- Once the cell is disrupted, proteolytic enzymes that were carefully packaged and controlled within intact cells are released, which can begin to degrade the proteins in the extract, including the protein of interest.
- To slow down unwanted proteolysis, all extraction and purification steps are carried out at 4 ° C, and a range of protease inhibitors are also included in the buffer.
- Common examples of inhibitors include diisopropylphosphofluoridate (DFP), phenylmethylsulfonylfluoride (PMSF), and tosyl phenylalanyl chloromethyl ketone (TPCK) (all serine protease inhibitors); iodoacetate and cystatin (thiol protease inhibitors); pepstatin (aspartic protease inhibitor); EDTA and 1,10-phenanthroline (metalloprotease inhibitors); and amastatin and bestatin (exopeptidase inhibitors).
3. Enzyme substrate and cofactors
- Low levels of the substrate are often included in extraction buffers when purifying an enzyme since binding the substrate to the enzyme’s active site can stabilize the enzyme during purification processes.
- Where relevant, cofactors that might otherwise be lost during purification are also included to maintain enzyme activity.
4. EDTA
- This may be present to remove divalent metal ions that can react with thiol groups in proteins to give mercaptides.
5. Polyvinylpyrrolidone (PVP)
- This is often added to extraction buffers for plant tissue.
- Plant tissues contain considerable amounts of phenolic compounds that can bind to enzymes and other proteins through non-covalent forces, including hydrophobic, ionic, and hydrogen bonds, resulting in protein precipitation.
- Therefore, insoluble PVP (which mimics the polypeptide structure) is added to adsorb the phenolic compounds which can then be removed by centrifugation.
- Thiol compounds (reducing agents) are also added to minimize the activity of phenol oxidases and thus prevent the formation of quinones.
6. Sodium azide
- For buffers that are to be stored for long periods of time, antibacterial and/or antifungal agents are sometimes added in low concentrations.
- Sodium azide is frequently used as a bacteriostatic agent.
Importance of an extraction buffer
- The main purpose of the lysis buffer is to isolate the compounds of interest and keep them in a stable environment.
- They are of enormous importance in practical biochemical work since many biochemical molecules are weak electrolytes and their ionic state varies with pH. Therefore, it is necessary to stabilize this ionic state during the course of a practical experiment.
