Separating Biological Macromolecules by Agarose Gel Electrophoresis.
Electrophoresis is a method of separating biological macromolecules like DNA and proteins from a mixture of similar molecules. An electric current is passed through a medium containing the mixture and each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. Agarose and acrylamide gels are the media commonly used for electrophoresis. In this experiment, we used agarose gel to act as a molecular sieve.
Theory.
There are many types of electrophoresis. I have listed 10 of them with their description below.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis): SDS is one of the most common types of electrophoresis. SDS is a detergent, which denatures proteins by binding to the hydrophobic regions and essentially coating the linear protein sequence with a set of SDS molecules. The SDS is negatively charged and thus becomes the dominant charge of the complex. The number of SDS molecules that bind is simply proportional to the size of the protein. Therefore, the charge to mass ratio should not change with size. In solution (water), in principle all different sized proteins covered with SDS would run through an inert polymer, polyacrylamide. The density and pore size of this polymer can be varied by just how you make it. Thus, the size of molecules that can pass through the matrix can be varied. This determines what molecular weight range the gel will have the highest resolving power.
Native Gels: It is also possible to run protein gels without SDS. These are called native gels in that one does not purposely denature protein. Here, the native charge on the protein (divided by its mass) determines how fast the protein will travel and in what direction.
Electrofocusing Gels: Another variation of gel electrophoresis is to pour a gel that purposely has a pH gradient from one end to the other.
