One of the most widely utilised analytical tools in molecular biology and disease diagnosis is the procedure and methods of electrophoresis. The principle of this procedure is that “electrophoresis is the migration of electrically charged particles or ions in solutions due to an applied electric field”1. The ability to separate substances which are almost identical including different proteins for analysis has increased throughout the past 7 decades.This is contributed to the introduction of zone electrophoresis in paper and more recently in gels like polyacrylamide and agarose. Today the methods used in electrophoresis have increased our advances in biochemistry, molecular biology and studies and diagnosis of disease, for example cancer and it has also become an invaluable tool in forensic science as it can identify species and individuals1.History and Background of Electrophoresis.
All though it was in 1791 when Faraday first presented his laws of electrolysis due to his experiments he formulated the theories of electrophoresis1. It was this procedure which was first pioneered in 1930 by a medical student working under Svedberg who had received the Nobel Prize for his own work on ultracentrifugation on proteins. The students name was Arne Wilhelm Kaurin Tiselius and it was then he published his thesis “Moving Boundary Electrophoresis” 2. He performed his experiments in a quartz U-tube using ultra violet light to photograph protein boundaries. However boundaries were often blurred in appearance caused by the heat in the solution, therefore results were not entirely accurate.It was not until 1937 he invented an electrophoresis apparatus which made it possible to obtain a much higher resolution and separation of charged molecules.
The first of his experiments which were carried out with horse serum enabled the globulins to separate into three parts. Tiselius named the three parts alpha, beta, and gamma. Further investigation showed that the three areas were different chemically and that the antibodies, or immunoglobulins, were found in the gamma globulin or between the beta and gamma globulins. Along with these set of experiments and the method he invented combined made moving boundary electrophoresis an accurate analytical method. All experiments and studies of this nature throughout the 1940s and 1950s were carried out using “Tiselius”-type apparatus equipped with Schlieren optics to visualize nucleic acid separations.The Development of Gel Electrophoresis. The first introduction of a gel electrophoresis was presented in 1950 by Gordon et al and was improved by Wieme by the introduction of the gel being supported on a glass slide. This cut out the need of the paper wicks thus preventing the adsorption of the voltage gradient2.
The next major step was in 1953 when the concept of crossed immuno electrophoresis in agar gels. This proved to be a huge landmark in separation techniques.It showed that not only could they separate the complex matrix but were able to also detect directly on the same plate. Thus the beginning of 2D electrophoresis, as not only did it have the zone electrophoretic step, but also by using the correct anti-sera would activate the immuno-detection step by diffusion.
The impact of this procedure was immense in biochemistry and molecular biology techniques.There was also a major impact in the study of disease for example the most famous Laurell’s Rocket technique1. This 2D technique showed to be a valuable tool for identification and quantification of proteins in an agarose gel. This procedure has been widely used in biomedical separations especially for the diagnosis of disease and the presence of antibodies in sera. Though these techniques are not as commonly used these days they did lay the foundations for western blotting techniques on 1D sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2D gels.The Introduction of Perfection.
It was during the 1960s that polyacrylamide gels were introduced as they were very efficient in molecular sieving and molecular filtration. Then in 1967 the introduction of sodium dodecyl sulphate (SDS) to the polyacrylamide. This proved to give a very good resolution of membrane proteins.
The SDS does have a disadvantage and that is it possesses a strong denaturing effect, although according to Vesterberg1 Hjerten and others showed that the hydrophobic membrane proteins showed less susceptibility to the denaturing effect of the SDS, than the hydrophilic proteins and were easily reactivated by adding an excess of neutral detergent. The SDS-PAGE has proved to be an important and valuable tool for the analysis and isolation of membrane proteins. This electrophoretic method is the most widely used in biomedical laboratories today.Estimation of protein size, assessment of the proteins purity, quantitation of proteins, monitor protein integrity, comparing composition of different samples, analysis of number and size of polypeptides, and for western blotting and also has a 2D capability. Throughout the 1970s techniques were changing and concepts of isotachophoresis and isoelectric focusing were being introduced, though the main aim throughout this decade was to find a way of miniaturising the equipment for analysis of certain biological samples4. Though this concept of smaller materials and the use of thin capillary like tubes was first introduced in 1970 it was not until the 80s capillary electrophoresis was introduced.
The idea of miniaturised equipment for certain samples example ribonucleotides came along well before modern capillary electrophoresis, it was in 1953 when fine silk fibres were used for micro electrophoresis to identify the ribonucleotides in single cells4. The effectiveness of capillary electrophoresis is very similar to a high performance liquid chromatography. This procedure took essential components from HPLC and electrophoresis and was more of an instrumental approach to electrophoresis.
Although capillary electrophoresis has a relatively short history it has proved to be very effective for any bioanalysis. In contrast to slab gel electrophoresis capillary electrophoresis takes advantage of two types of driving force, the force causing the electrophoretic migration and the force caused by the electoosmotic flow through the capillary.It is comprised of fused silica capillary’s 20-100 ?m in diameter and 20-100 cm long for the separation channel and is encased in a temperature-controlled cartridge. The inlets and outlets are placed in the sample and the buffer vial. Injection is by pressure or electrokinetic means.
A sample vial is temporarily pressurised to allow the flow of sample into the capillary in the pressure method and the electrokinetic method uses the electric field to migrate the charged particles into the capillary4.