For the separation of particular protein of interest, there are different methods that are used that how proteins are separated in a mixture. Two methods for the separation of proteins are:
• Selective (specific to proteins, biochemical binding).
• Non-selective (it involves mass and net charge, on the basis of these properties proteins are separated)
Electrophoresis: a non-selective approach is defined as a molecule with a net charge will move in an electric field. For example, any charge molecule subject to an electric field will move.
Electrophoresis depends on:
• Net charge of protein
• Applied electric field
To conduct electrophoresis we use gels. There are many differences between these two gels:
i. Agarose gel:
• Used for larger molecules.
• It does not identify small differences between two molecules bands).
• Used for the separation of DNA.
• It contains large pores.
• Ethidium bromide is used to visualize agarose gel.
ii. Polyacrylamide gel
• It is used for smaller molecules.
• It identifies the differences between two molecules (bands).
• It is used for DNA and protein separation.
• It contains small fine pores.
SDS-PAGE: a non-selective method of electrophoresis
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique of electrophoresis (a molecule with a net charge will move in an electric field) in which protein molecules are separated on the basis of mass. SDS-PAGE is widely used in molecular biology, genetics, proteomics, forensics and biochemistry.
What we do in SDS-PAGE?
• The mixture of protein is first dissolved in a solution of SDS.
• SDS is an anionic detergent that disturbs nearly all non-covalently interactions in proteins. Moreover, it is a denaturing agent that is used to denature protein and the denaturing agent which is used to denature protein is Urea.
• To make protein linear we have to remove disulphide bonds for this we will use Mercaptoethanol.
• SDS-protein complex has a large net negative charge that is proportional to mass of the protein. The negative charge acquired on binding SDS is usually much greater than charge on the negative protein.
• The SDS-protein complexes are then subjected to electrophoresis.
• Two residues of molecules contain one SDS-PAGE.
Visualization of protein
• For the visualization of proteins Coomassie Brilliant Blueor silver staining is used.
• We can also radio-label, but it's very expensive and dangerous.
Separation on the basis of mass. We can achieve sieving effect (sieving effect depends on pore size) through gels. Small proteins easily pass on from the pores; some proteins will not, some find alternative path on the basis of mass. Pore size depends on gel concentration. More is the concentration of gel, smaller the size of pore.
Agent responsible for cross linkage
Methylene bis acrylamide is an agent that is used to produce cross linkages. Acrylamide polymerizes the gel. Benefit of cross linkage is to pass large molecules easily from the pores.
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