Electrophoretic technique is used to separate the charged particles on the basis of their mass/molecular weight. There are many types like capillary electrophoresis, 2d electrophoresis. Based on the gel matrix used there are two types of electrophoresis one uses polyacrylamide gel and the other uses is agarose gel . Though there are many types of electrophoresis which uses different suspending materials like starch grain, cellulose acetate, paper but the main two are polyacrylamide and agarose gel electrophoresis. Polyacrylamide gel electrophoresis is also two types one is native (Native-PAGE) and the other is Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (sds-page gel). Native-PAGE is used to separate molecules in their native stage without denaturing the 3D structure while SDS-PAGE is used to separate all types of subunits as well as complete molecules. In SDS-PAGE sodium dodecyl sulfate is used as a anionic detergent which provides negative charge to the molecules to be separated.
Electrical field in the electrophoresis
Many biological molecules like proteins, nucleic acids carry electrical charge, the magnitude of which depends on the pH, and composition of the suspending medium and also on the particular molecule. These charged particles/molecules migrate in the solution to the electrode of opposite charge (polarity) in the presence of electrical field. If the molecule of charge q is present in an electric field of strength x then the force on the molecule causing it to move is qx. this force is balanced by the frictional resistance f to give a terminal velocity v.
Thus qx = fv
Now according to stoke's law of viscocity;
f = 6Πηr
thus qx = 6Πηrv
the mobility of the molecule thus depends on the viscocity of the medium and the size, shape of the molecule and also on the charge, the molecule contains.
The suspending media need to be selected carefully since some buffer ions may react with the compound to be absorbed as for example borate forms complex with sugar, citrate/EDTA will chelate metal ions of some enzymes. The pH must be chosen in such a way that it does not affect the protein/sample under investigation to be precipitated in the solution before elelctrophoresis
A stable DC source of electricity is required which can provide constant current or voltage. 5-8V/cm field strength is suitable for most of the separations at room temperature. If the field strength becomes greater than 10V/cm there will be heating, heat will be generated on the gel causing damage to the sample as well as gel because there will be disturbance in zone caused due to lost of water by evaporation concentrating the buffer. Though there are some methods of cooling but sophisticated equipments are required.
SDS-PAGE Gel Visualization
The different protein bands are visualized with the help of staining solution. Different proteins are separated based on the molecular weight and total charge contained within the proteins.
Gel is prepared by the use of acrylamide and small amount of crosslinker methylene bisacryl amide in the presence of ammonium persulfate as a catalyst. the tetremethylethylenediamine (TEMED) is also added which creates the persulfate free radicals from ammonium persulfate which initiates the polymerization. The pore size of the gel is determined by the various concentration of monomers. The acrylamide is toxic (neurotoxic) and must be handled with care.