Electrophoresis types and applications
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Types and advancements of Electrophoresis
2.1 Paper electrophoresis
Paper electrophoresis is one of the simplest methods of electrophoresis. The sample is applied onto a point of a strip of filter paper that has been moisturized with a buffer solution. Each end of the strip is then dipped into separate tanks containing the buffer solution and a different electrode (anode or cathode). An electric current is then applied and the sample will then move towards the electrode with the opposite polarity. When the process is done, the strip is then dried and viewed with a detection system.
Paper electrophoresis has often been compared to paper chromatography due to their similarity in their mode of action. However, chromatography separates a sample based on its polarity while paper electrophoresis separates the sample based on its charge by applying a running electrical charge from one terminal to the other .
2.2 Agarose gel electrophoresis
Agarose is a polysaccharide that forms pores with sizes ranging from 100 to 300 nm in diameter. The size of the pore correlates with the concentration of the agarose gel. The higher the concentration of the agarose gel the smaller the pore size and vice versa. Agarose gel electrophoresis is often used to separate DNA or RNA fragments of different length. It involves the movement of negatively charged DNA or RNA molecules from the negative electrode to the positive electrode. The molecules are separated based on their molecular size.
2.3 Polyacrylamide gel electrophoresis (PAGE)
There are two types of polyacrylamide gel namely the dissociating and non-dissociating gels. A non-dissociating gel separates the proteins in their native form to conserve the protein structures, functions and activity. It is useful when the protein of interest is wanted at the end of the procedure for subsequent experiment. A dissociating gel denatures the protein into its constituent polypeptides to determine the polypeptide composition of the sample. Native gel electrophoresis is a non-denaturing gel that has a higher resolving power than the SDS-PAGE when used for protein separations.
Polyacrylamide gel is made up of chains of acrylamide monomers (CH2=CH-CO-NH2) that is cross linked with N, N′-methylenebisacrylamide units (CH2=CH-CO-NH-CH2-NH-CO-CH-CH2), also commonly known as “bis”. The concentration of bis shall determine the pore size of the polyacrylamide gel. Due to its higher resolving power than agarose gel electrophoresis, polyacrylamide gel is most commonly used in examining proteins and families of chain-terminated DNA molecules. Additionally, native polyacrylamide gel is capable of separating DNA molecules with a size difference of one nucleotide, such as in single nucleotide polymorphism (SNP) studies.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a rapid method for quantifying and characterizing protein or small molecular weight peptides. SDS-PAGE separates proteins based on their molecular weight. There are two variants of the polyacrylamide gel namely the gradient and SDS-Urea gels. Gradient gels are often employed to separate proteins in just a single resolving gel without the need of stacking gel. SDS-Urea gels are used when the charge of the proteins is significantly similar to the mass of the protein, such as membrane proteins and immunoprecipitates . Solubilization, denaturation and dissociation of the polypeptides chains without altering the proteins intrinsic charge is achieved with use of urea.
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