The composition of buffer used for extraction is an essential part for disrupting cell to release its components. It consists of buffer salts and ionic salts to maintain ionic strength and osmolarity of lysates. Detergents present breaks up or disrupt the membranes of cells and release its components.
Common components of recovery buffer includes antioxidants, EDTA, enzyme substrate and cofactors, enzyme inhibitors, sodium azide, Polyvinylpyrrolidone etc.
Cellular components are usually in highly reduced environment so when they are released on disruption of cells, they get exposed to oxidized environment hence, reducing agents such as dithiothreitol, β mercaptoethanol, cysteine or reduced glutathione are added to the buffer which prevents oxidation of sulphydryl groups of proteins to form inter- and intramolecular disulphide bond.
Proteins and enzymes of cell are released during disruption/ lysis of cell. Hydrolytic enzymes such as lysozymes also gets released which degrade the protein of interest along with other proteins present in extract. In order to slow down unwanted proteolysis, all extraction and purification steps can be carried out at 4oC.
On other hand, various protease inhibitors can also be used which should be selected in such a way that they shouldn’t inhibit enzymes responsible for cell lysis. Each inhibitor is specific for a particular type of protease.
Common examples of the enzyme inhibitors are phenylmethylsulphonylfluoride (PMSF), pepstatin, EDTA, di-isopropylphosphofluoridate (DFP), cystatin, iodoacetate etc.
Enzyme Substrate and Cofactors
Enzyme substrate at low level are often added to extraction buffer. The binding of substrate to active site of enzyme is very specific so, this binding stabilizes the enzyme during extraction and purification process. Various enzymes specific cofactors are also added into it to maintain enzyme activity which otherwise might be lost during recovery process.
Ethylenediaminetetracetic acid (EDTA)
EDTA, a chelating and stabilizing agent is added in extraction buffer to remove divalent meal ions that can react with thiol groups of protein giving mercaptids.
PVP is often added to extraction buffer for plant tissues. Plant tissues contain considerable amount of phenolic compound that can precipitate proteins. Insoluble PVP is added to adsorb these phenolic compound which can then be removed by centrifugation. Other than PVP, thiol compound can also be added to minimize their activity.
It does not have a specific function during extraction and purification process. Basically, it is an antibacterial and/or antifungal agent which is used as a preservative for buffer. It prevents spoilage of extraction buffer if it needs to be stored for long period of time. Low concentration of sodium azide is added to buffer which prevents bacterial and fungal contamination.