Electrophoresis: Types, Principles, and Applications

Electrophoresis is a technique used to separate charged molecules like DNA, RNA and Proteins using an applied electric field. the separation can be based on size or charge. The molecules will migrate towards the oppositely charged electrode through a gel made of agarose or acrylamide. this technique is very useful when you need to purify your DNA, RNA, or your Protein from a mixture. Most of them are charged because they have ionizable functional groups. So, they can easily be separated using electrophoresis

The gel acts as a sieve. Smaller molecules move faster and larger molecules move slower.

Types of Electrophoresis

1. Agarose Gel Electrophoresis
   • used in separation of– DNA
   • separation based on– size
   • type of gel– agarose- extracted from sea weed (available in powdered form). once it is mixed with water, heated, and cooled, it forms a gel.
     • 0.7-1% agarose are used when you want to separate large fragments
     • 2-3% gels for smaller fragments
   • Buffer used– Tris Acetate EDTA (TAE) and Tris Borate EDTA (TBE) to maintain the pH and conduct electricity
   • Visualized by– ethidium bromide
   • Applications
     • Estimate the size of DNA molecules by running the sample alongside a ladder or marker
     • Genetic fingerprinting
   • Advantages
     • Easier to prepare as it contains a single component
     • Samples do not denature
       
2. Polyacrylamide Gel Electrophoresis
   • Used in separation of– Protein
   • Separation based on– electrophoretic mobility which depends on shape, size, and charge. PAGE can be of 2 types- Native and Sodium Dodecyle Sulphate (SDS PAGE). In SDS PAGE, the sample proteins are denatured so they all have a negative charge at the start of electrophoresis. So, in the case, separation is purely based on molecular weight. In Native PAGE, it is not the case.
   • Gel– Polyacrylamide. It is a complex gel made of mainly two components- acrylamide (polymers of which form the gel) and N,N’-methylenebisacrylamide for cross linking). The entire PAGE gel consists of a resolving gel (below) and a stacking gel (above). The stacking gel ensures the proteins start migration at the same point.
   • Buffer used– Tris Glycine SDS
   • Visualized by- Protein specific dye or coomasie blue
   • Applications
     • Estimate protein size
     • Preliminary step to western blotting
     • Estimate purity of protein
   • Advantages
     • Gel is highly stable
     • Greater resolving power
       
3. Pulse field Electrophoresis
   • Used in separation of– very large DNA molecules (15-20 kB)
   • Separation based on– size and how quickly they can change direction. The electric field alternates in direction giving a zig zag pattern. Larger fragments take time to change direction.
   • Gel– Agarose (0.5-1.5%)
   • Buffer used– Tris Borate EDTA (0.5 X TBE) or Tris Acetate EDTA (1%TAE)
   • Visualized by– ethidium bromide
   • Applications–
     • Bacterial genome mapping
     • Yeast karyotyping
   • Advantages–
     • Can separate large DNA’s
     • Universal method for subtyping of bacteria
       
       

Now that you are familiar with the basic principle behind different types of electrophoresis, a related post where I talk about the instrumentation of each type of electrophoresis is dropping soon.