Protein purification: Protein purification is a series of processes used to isolate a specific type of protein from a complex mixture. Protein purification is important for the characterization of the function, structure and interactions of the protein of interest. The various steps in the purification process may include the isolation of the protein from a matrix, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of the proteins depends on protein size, physio-chemical properties, binding affinity and biological activity (Burgess. 2008).
Chromatographic methods
• Ion-exchange chromatography separates proteins based on charge. Columns can either be prepared for anion exchange or cation exchange. Elution of the target proteins is done by changing the pH in the column, which results in a change or neutralisation of the charged functional groups of each protein (www.biotech.about.com).
• Size-exclusion chromatography (gel filtration) separates larger proteins from small ones, since the larger molecules travel faster through the cross-linked polymer in the chromatography column. The large proteins do not fit into the pores of the polymer whereas smaller proteins do, and take longer to travel through the chromatography column, via their less direct route.
• Affinity chromatography is a very useful technique for completing the protein purification process. Beads in the chromatography column are cross-linked to ligands that bind specifically to the target protein. The protein is then removed from the column by rinsing with a solution containing free ligands. This method generally gives the purest results and highest specific activity compared to other techniques (Wilson and Walker. 2006).
Blotting: is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose, polyvinylidine fluoride (PVDF) or nylon membrane.
Electrophoresis
Electrophoresis is the main biochemical technique for molecular separation. Electrophoresis can be one dimensional or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).When the detergent SDS (sodium dodecyl sulfate) is used with proteins, C all of the proteins become negatively charged by their attachment to the SDS anions. When separated on a polyacrylamide gel, the procedure is known as SDS—PAGE (Sodium Dodecyl Sulfate- PolyAcrylamide Gel Electrophoresis). The technique has become a standard means for molecular weight determination (Lodish et al. 2004).